Osteogenesis Imperfecta Proteomic Characterisation Extracellular Vesicles
Osteogenesis Imperfecta (OI), known as ‘brittle bone disease’, is a rare heritable debilitating connective tissue disease, with incidence of approximately 1 in 20,000 birth. Majority of the cases are autosomal dominant in inheritance occurring due to mutations in genes encoding the α-chains α1 and α2 of type 1 collagen, COL1A1 and COL1A2 genes respectively. Recessive osteogenesis imperfecta also occurs but its rare and more lethal than the autosomal dominant form. It is caused by mutations in genes coding for proteins that interact with type 1 collagen and affect its processing, post-translation modification and secretion. Because of collagen’s vital role in the musculoskeletal system, OI patients often present with bone fragility manifesting as multiple fractures, skeletal deformities and growth delay.
The current gold standard for managing the manifestations of this bone fragility is by re-alignment of the bones with osteotomy then internal fixation with an intra-medullary nail. However, this management modality is far from ideal, as the risk of delayed healing and non-union at the osteotomy site ranges from 35-50%, depending on the involved bone. These complications often require a complex revision surgery and; therefore, result in an increased burden on the patient, family and the health care institution. Therefore, the need for a method that decreases the risk of these complications in children with OI is still unmet. To address these bone deficiency complications, several treatment options have been assessed. Autologous bone grafts are known for their strong osteogenic abilities with minimum risk of immune-rejection or disease transmission.
However, this treatment modality has several drawbacks such as limited availability and serious complications of the harvesting procedure. Bone allografts are an alternative but they are less osteogenic and have the risk of immune-rejection as well as disease transmission. Because of this still unmet need for adequate safe bone regeneration, regenerative medicine is now looking at extracellular vesicles as a solution to this problem.
Extracellular Vesicles (EVs) is a term that collectively refers to heterogeneous group of membrane-bound vesicles secreted by cells into their extracellular environment, ranging in size from few nanometers to few micrometers. In the human body, they can be isolated from various bodily fluids including blood, urine, saliva, breast milk and cerebrospinal fluid. This finding has increased EVs’ practicality for easy access to further study them and explore their role in the healthy and the diseased human body. Thus far, researchers defined at least 3 main subgroups of EVs: 1) exosomes, 2) apoptic bodies and 3) cellular microparticles/microvesicles/ectomes. Each subgroup has its unique biogenesis, function and characteristic. Unfortunately, there is a lack of consensus on identifying biomarkers to distinguish these subgroups. In addition, different methods by which the EVs are isolated result in variable composition of EVs and subfractions of EV subgroups. For the aforementioned reasons, to unify the nomenclature and minimize the confusion, we will from here onward refer to all types of vesicles in our study as EVs. EVs have been shown to carry proteins, lipids, coding and non-coding RNAs, with the specific content depending on the secreting cell and its metabolic status at the time of secretion. Carrying such valuable content, EVs derived from MSCs In the context of tissue repair, it has been shown that mesenchymal stem cells exert their function in a paracrine manner.
In addition to this vital discovery, researchers have also identified EVs to be the active components of these paracrine functions of MSCs. Interestingly also, osteoblasts, which are the bone-forming derivatives of MSCs, have been shown to exert their function in bone in paracrine manner via EVs also. EVs are gaining wide attractions from researchers for there regenerative potentials, particularly those derived from mesenchymal stem cells. For instance, MSC-EVs have enhanced neurogeneration post-stroke in one study, as well as cardiac tissue regeneration in another. However, MSC-EVs role in bone regeneration is at an early stage of investigation but the preliminary studies done on this topic are quiet promising. MSC-EVs have been shown to regulate osteoblast activity and differentiation in-vitro. This was proven by an increased expression of osteogenic genes as well as obvious calcium deposits with Alizarin staining. In-vivo, MSC-EVs have promoted bone formation in several animal models, further proving the capability of EVs to induce osteogenic differentiation of MSCs.
One study reported that human embryonic MSC-derived exosomes promoted osteochondral regeneration in a rat model of critical-sized osteochondral defect, whilst another study has used bone marrow MSC-derived exosomes to promote femoral fracture healing in a CD9 null mice (a strain that is known to produce reduced levels of exosomes). These interesting findings have prompted other researchers to analyze and study EVs secreted by osteoblasts. It was found that EVs derived from MC3T3-E1 mouse calvarial pre-osteoblasts, which reside in mineralized extracellular matrix, are capable of inducing osteogenic differentiation of bone marrow stromal cells. Because of our interest in Osteogenesis Imperfecta and the difficulties that are faced in its management, we wanted to explore the paracrine behavior of OI-derived mesenchymal stem cells and their derived osteoblasts, as well as, the possible role of the secreted EVs in the pathophysiology of this disease and its complications. Given that autosomal dominant OI with mutations in COL1A1 and COL1A2 genes is the most prevalent, we will be conducting our experiments with MSCs derived from OI patients with this genotype.
