Occurrence and Treatment of Bone Atrophic Non-Unions Investigated by an Integrative Approach

Recently developed atrophic non-union models are a good representation of the clinical situation in which many nonunions develop. Based on previous experimental studies with these atrophic non-union models, it was hypothesized that in order to obtain successful fracture healing, blood vessels, growth factors, and (proliferative) precursor cells all need to be present in the callus at the same time. This study uses a combined in vivo-in silico approach to investigate these different aspects (vasculature, growth factors, cell proliferation). The mathematical model, initially developed for the study of normal fracture healing, is able to capture essential aspects of the in vivo atrophic non-union model despite a number of deviations that are mainly due to simplifications in the in silico model. The mathematical model is subsequently used to test possible treatment strategies for atrophic non-unions (i.e. cell transplant at post-osteotomy, week 3). Preliminary in vivo experiments corroborate the numerical predictions. Finally, the mathematical model is applied to explain experimental observations and identify potentially crucial steps in the treatments and can thereby be used to optimize experimental and clinical studies in this area. This study demonstrates the potential of the combined in silico-in vivo approach and its clinical implications for the early treatment of patients with problematic fractures

Multiscale Modeling of Bone Healing

Bone is a living part of the body that can, in most situations, heal itself after fracture. However, in some situations, healing may fail. Compromised conditions, such as large bone defects, aging, immuno-deficiency, or genetic disorders, might lead to delayed or non-unions. Treatment strategies for those conditions remain a clinical challenge, emphasizing the need to better understand the mechanisms behind endogenous bone regeneration. Bone healing is a complex process that involves the coordination of multiple events at different length and time scales. Computer models have been able to provide great insights into the interactions occurring within and across the different scales (organ, tissue, cellular, intracellular) using different modeling approaches [partial differential equations (PDEs), agent-based models, and finite element techniques]. In this review, we summarize the latest advances in computer models of bone healing with a focus on multiscale approaches and how they have contributed to understand the emergence of tissue formation patterns as a result of processes taking place at the lower length scales

Towards a New Spatial Representation of Bone Remodeling

Irregular bone remodeling is associated with a number of bone diseases such as osteoporosis and multiple myeloma. Computational and mathematical modeling can aid in therapy and treatment as well as understanding fundamental biology. Different approaches to modeling give insight into different aspects of a phenomena so it is useful to have an arsenal of various computational and mathematical models. Here we develop a mathematical representation of bone remodeling that can effectively describe many aspects of the complicated geometries and spatial behavior observed. There is a sharp interface between bone and marrow regions. Also the surface of bone moves in and out, i.e. in the normal direction, due to remodeling. Based on these observations we employ the use of a level-set function to represent the spatial behavior of remodeling. We elaborate on a temporal model for osteoclast and osteoblast population dynamics to determine the change in bone mass which influences how the interface between bone and marrow changes. We exhibit simulations based on our computational model that show the motion of the interface between bone and marrow as a consequence of bone remodeling. The simulations show that it is possible to capture spatial behavior of bone remodeling in complicated geometries as they occur \emph{in vitro} and \emph{in vivo}. By employing the level set approach it is possible to develop computational and mathematical representations of the spatial behavior of bone remodeling. By including in this formalism further details, such as more complex cytokine interactions and accurate parameter values, it is possible to obtain simulations of phenomena related to bone remodeling with spatial behavior much as \emph{in vitro} and \emph{in vivo}. This makes it possible to perform \emph{in silica} experiments more closely resembling experimental observations.Comment: Math. Biosci. Eng., 9(2), 201

The Role of Osteocytes in Targeted Bone Remodeling: A Mathematical Model

Until recently many studies of bone remodeling at the cellular level have focused on the behavior of mature osteoblasts and osteoclasts, and their respective precursor cells, with the role of osteocytes and bone lining cells left largely unexplored. This is particularly true with respect to the mathematical modeling of bone remodeling. However, there is increasing evidence that osteocytes play important roles in the cycle of targeted bone remodeling, in serving as a significant source of RANKL to support osteoclastogenesis, and in secreting the bone formation inhibitor sclerostin. Moreover, there is also increasing interest in sclerostin, an osteocyte-secreted bone formation inhibitor, and its role in regulating local response to changes in the bone microenvironment. Here we develop a cell population model of bone remodeling that includes the role of osteocytes, sclerostin, and allows for the possibility of RANKL expression by osteocyte cell populations. This model extends and complements many of the existing mathematical models for bone remodeling but can be used to explore aspects of the process of bone remodeling that were previously beyond the scope of prior modeling work. Through numerical simulations we demonstrate that our model can be used to theoretically explore many of the most recent experimental results for bone remodeling, and can be utilized to assess the effects of novel bone-targeting agents on the bone remodeling process

Multiscale Modeling of Bone Healing: Toward a Systems Biology Approach

Bone is a living part of the body that can, in most situations, heal itself after fracture. However, in some situations, healing may fail. Compromised conditions, such as large bone defects, aging, immuno-deficiency, or genetic disorders, might lead to delayed or non-unions. Treatment strategies for those conditions remain a clinical challenge, emphasizing the need to better understand the mechanisms behind endogenous bone regeneration. Bone healing is a complex process that involves the coordination of multiple events at different length and time scales. Computer models have been able to provide great insights into the interactions occurring within and across the different scales (organ, tissue, cellular, intracellular) using different modeling approaches [partial differential equations (PDEs), agent-based models, and finite element techniques]. In this review, we summarize the latest advances in computer models of bone healing with a focus on multiscale approaches and how they have contributed to understand the emergence of tissue formation patterns as a result of processes taking place at the lower length scales

The role of neurohumoral modulation in fracture healing:lifting a tip of the veil

The process of fracture healing is an extremely complex process in which an immense amount of biological factors from the musculoskeletal-, neurological-, vascular- and immune-system interact with each other. In this thesis, we aimed to evaluate the influence of some of these factors on the fracture healing process. The most important questions of this thesis are: 1. Does concomitant traumatic brain injury enhance the fracture healing process? 2. How does the blockage of the neurotransmitter substance P influence fracture healing? 3. Is the CatWalk gait analysis system suitable for gait analyses in small animal fracture models? 4. Does the arginine-nitric oxide metabolism in spongious bone grafts influence the outcome of non-union treatment? 5. What is the impact of plasma-derived micro-vesicles on the viability and proliferation of osteoblasts in a small animal fracture model? 6. Are there changes in prevalence, activity, and functionality of circulatory and pulmonary neutrophils after an intramedullary stabilized femoral fracture in a small animal fracture model

Development of a clinically relevant strategy to promote fracture healing in an atrophic non-union model using mesenchymal stem cells

Atrophic non-union is a major complication following fracture of a bone. It represents a biological failure of the fracture healing process and occurs in 5-10% of cases. A number of factors predispose to atrophic non-union including high energy injuries, open fractures, diabetes, and smoking. Atrophic non-unions cause immense patient morbidity and consume large amount of health care resources. Bone grafts taken from the iliac crest contain biologic components required for fracture healing and are considered as the gold standard treatment of aseptic atrophic non-union. However, harvesting bone grafts from the iliac crest is associated with significant patient morbidity which can reduce quality of life. Mesenchymal stem cells (MSCs) have the ability to proliferate and undergo multilineage differentiation. The emergence of MSC therapy provides an alternative strategy for treating impaired fracture healing. MSCs contribute to normal fracture healing both directly as bone progenitor cells and indirectly as mediator secreting cells. Although a number of studies have shown that MSCs can promote bone regeneration in small animal fresh critical size defects, this is not analogous to most clinical aseptic atrophic non-unions which do not have a significant bone gap. There remains therefore a clinical need for an appropriate strategy for using stem cells in atrophic non-unions. Thus, the aim of this study aim was to develop a clinically relevant strategy to promote fracture healing in an atrophic non-union model using the percutaneous injection of MSCs as a minimally invasive technique. An atrophic non-union model was established and validated. A small (1 mm) non-critical size defect was created at the mid shaft tibia and the fracture site was stabilised using an external fixator. Atrophic non-union was induced by stripping the periosteum for one bone diameter either side of the osteotomy site and curettage of the intramedullary canal over the same distance. The procedure reliably created an atrophic non-union. Fracture healing was evaluated using (1) serial radiography, (2) micro-computed tomography, (3) histomorphology and (5) biomechanical testing. Fracture scoring systems including the radiographic union scale in tibia (RUST) and the Lane & Sandhu score were validated in a preclinical model. A simple sample preparation technique for evaluating bone mechanical properties was developed and used to assess the stiffness and strength of the fracture repair. Percutaneous injection of MSCs locally into the fracture site in the early ‘post-injury’ period at three weeks after induction of atrophic non-union was found to improve the fracture healing process significantly (83% of cases), while MSCs implantation in the late ‘post-injury’ period at eight weeks after induction of atrophic non-union showed no significant improvement of fracture healing (20% of cases). Percutaneous local implantation of MSCs rescued the fracture healing process in cases destined to progress to atrophic non-union. In clinical practice, there may be an advantage using MSCs from a universal donor as the processes of MSC isolation and preparation are expensive and time consuming. To investigate the feasibility of using non-autologous cells, the atrophic non-union was used to determine the bone regenerative potential of using xenogeneic donor hMSCs in an atrophic non-union. The results demonstrated that the therapeutic effect of using hMSCs in a xenogeneic manner to promote fracture healing in the rat atrophic non-union model was comparable with rMSCs (88% of cases in both hMSCs and rMSCs) and there were neither significant clinical adverse effects nor adverse immune responses with the xenogeneic transplantation. However, MSCs did not persist at the fracture following injection. Perivascular stem cells (PSCs) taken from adipose tissue, which is an expendable source, have advantages over conventional MSCs as they are a defined and homogenous population and can be used without culture expansion. The administration of PSC using percutaneous injection improved the fracture healing process in atrophic non-union (60% of cases). This suggested that PSCs may present an appropriate choice for use in cell therapies to promote fracture healing in atrophic non-union. The results from this thesis can be applied to the development of a clinically relevant strategy using MSCs as a minimally invasive technique to promote fracture healing in atrophic non-union, in particular (1) the effectiveness of a cell therapy is likely to be highly dependent of the timing of injection relative to the stage of fracture healing, (2) hMSCs were as effective as rMSCs in promoting fracture healing, suggesting that it may be feasible to use an allogeneic strategy in humans, (3) the injected MSCs were not detectable even in case of successful repair, suggesting that they may act through a paracrine effect and (4) PSCs isolated from adipose tissue contributed to fracture healing in the atrophic non-union model, suggesting that adipose tissues can be used as an alternative cell sources for bone repair

Clinical and laboratory studies of mesenchymal stem cells in long bone fracture nonunion

Treatment of atrophic long bone fracture nonunion is challenging with current therapeutic interventions including bone morphogenetic proteins (BM Ps) or autologous mesenchymal stem cells (MSCs). In this work it was hypothesised that, total MSC numbers and their responsiveness to BMPs in the nonunion setting were compromised, leading to poor healing. Additionally, the rationale for systemic injection of MSCs to repair bone is based on a contentious concept of their widespread circulation. To address the number and functional competence of iliac crest bone marrow MSCs in nonunion, this study employed colony forming unit fibroblast and osteoblast assays (CFU-F, CFU-O) and flow cytometry enumeration of the CD45lowCD271+ cell population, to compare nonunions (n=11) with united long bone fracture patients (n=11). Unexpectedly, total number of MSCs, was higher in nonunion; however their proliferative capacity, was lower. No response to BMP-7, assessed by CFU-F, CFU-O and calcium deposition assays, was found in both nonunion and union study groups. Possible mechanical translocation of MSCs into the venous circulation was investigated using matched antecubital venous blood from the upper limb (UL) and lower limb femoral venous blood (LL) samples from nonunion patients undergoing reaming with reamer irrigation aspiration (RIA) (n=12) and other non-reaming procedures (NR, n=12) with control groups including UL from early rheumatoid arthritis (RA, n=11) and healthy controls (n=12). CFU-F assay results revealed the presence of MSC colonies in LL at higher frequencies than UL samples in RIA and NR, (8LL, 2PB) and (SLL, 1 PB) respectively. None were detected in the UL of RA and controls. Altogether, these results indicate a functional defect in proliferative capacity of MSCs in nonunion. MSCs however are unlikely to circulate and contribute to reduced healing at fracture sites. This points towards a generalized systemic effect of the nonunion state on MSC dynamics, which should be further explored in future