Original Full Length ArticleVascular development during distraction osteogenesis proceeds by sequential intramuscular arteriogenesis followed by intraosteal angiogenesis☆
Highlights
► We examined the contribution of the vascular network in surrounding muscle compartments to distraction osteogenesis. ► The period of active distraction was characterized by arteriogenesis in the surrounding muscle. ► During consolidation, angiogenesis predominated in the intraosteal region. ► Vessel formation proceeded from the surrounding muscle into the regenerate.
Introduction
Bone formation is intimately dependent on formation of vascular tissue. Inadequate blood supply is a major cause of delayed bony union and nonunion, leading to many of the complications in various post-surgical orthopedic treatments [1], [2]. Studies of human tibial fractures have shown impaired rates of healing as high as 46% when the patient had concomitant vascular injuries [3]. Fracture healing is also greatly compromised in situations of poor muscular coverage, of loss of muscular coverage due to extensive trauma, and of associated compromise of the vascular bed [3], [4]. Surgical transfer of tissue inclusive of the surrounding muscle is commonly used to salvage extensively traumatized lower extremities, and successful outcomes are generally credited to the maintenance of undamaged vascular beds in the transferred muscle tissue [4]. In experimental studies of fracture healing, development of lower-limb ischemia, in which the surrounding vasculature is damaged, has been also shown to lead to delayed union [5]. Such findings suggest therefore that the integrity of the vascular bed within the muscular compartment is essential to bone regeneration; however, the role that the surrounding vasculature plays in the morphogenesis of the vessels in the bone is not well defined.
Vascular morphogenesis during embryological skeletogenesis occurs by collateralization of vessels from the surrounding musculature into the newly forming bones and by subsequent formation of vascular tissue via angiogenesis concurrently with formation of both cortical and trabecular bone [6], [7], [8]. However, little is known about vascular morphogenesis during post-natal skeletal regeneration after injury or surgery. A basic understanding of the mechanisms by which vascular tissue growth is patterned is essential to developing an understanding of how vascular and skeletal cells coordinate the presentation of specific morphogenetic signals to each other within developing or regenerating bone tissues. Such an understanding is also important for maximizing the success of surgical procedures for bone repair, for tissue engineering of materials that will present morphogens in a specific spatiotemporal manner, for the development of scaffolds for cell-based therapies, and for determining the optimal timing of the application of therapeutic compounds that seek to promote vascular and/or bone healing [9].
Distraction osteogenesis (DO) represents an excellent orthopedic model to study both the processes of vascularization and the functional interactions between vascular and bone formation in a post-natal context. DO is a true regenerative process in which new bone formation is stimulated by mechanical stretching of the callus that forms following resection osteotomy [10], [11]. DO is used to lengthen and reshape bones and also to correct pseudarthroses that remain refractory to other forms of treatment [12], [13], [14]. The “tension stress” that is applied to the callus, or regenerate, appears to stimulate regulatory mechanisms that inhibit cartilage formation [15], [16], and these same signals appear to also promote extensive amounts of vascularization. Previous studies from our laboratory that have examined the functional relationship between vascular morphogenesis and bone tissue morphogenesis using a murine model of DO have shown that genes associated with vascular tissue formation were induced during each round of distraction [17]. Interestingly, the extent of vascularization has been proposed to provide many of the biological signals that direct differentiation of skeletal progenitor cells to the osteogenic lineages [18]. Consistent with this idea, we have shown that inhibition of formation of vascular tissue via antibody blockade of VEGF-receptor activity led to decreased bone formation and BMP2 expression and increased formation of cartilage and fibrous tissue [19]. Our recent findings also show that vascular smooth cells and endothelial cells are a major source of BMP2 expression during DO induced bone formation [20].
We hypothesized that the vascular network in surrounding muscular compartments would contribute to vascular formation and bone regeneration within the distraction regenerate. We tested this hypothesis by quantifying the temporal and spatial morphogenesis of vessel and bone formation across the time-course of DO in both muscular and osseous compartments. During the course of carrying out these studies, we developed and validated image-registration and image-subtraction procedures for micro-computed tomography (μCT) scans performed sequentially before and after demineralization on specimens containing contrast-enhanced vascular casts. Using these new techniques of contrast-enhanced, micro-computed tomography in conjunction with immunohistology, we specifically examined the contributory roles of arteriogenesis (formation of large vessels) and angiogenesis (formation of small vessels) to overall vascular tissue formation in both the surrounding muscular tissues and in the osseous regenerate during DO.
Section snippets
Study design
All animal studies were carried out under a protocol that was approved by the Institutional Animal Care and Use Committee (IACUC) at the Boston University School of Medicine. Male, C57BL/6J wild type mice were obtained from Jackson Labs (Bar Harbor, ME). All mice enrolled in the study weighed 25–35 g and were between ages 9 and 11 weeks of age at the time that they underwent DO surgery on the left femur. The total time-course for the experiment was 31 days. The distraction protocol consisted of
Development of μCT imaging methods for the concurrent analysis of vascular and mineralized tissues
Current approaches using contrast-enhanced vascular casts and μCT to visualize vessel networks within bone organs, particularly in the context of a bone injury, do not allow clear discrimination between vessels and bone tissue at the image resolution typically afforded by desktop μCT systems. The approach that was developed in this study was to image the same specimen before and after demineralization, to co-register the two 3-D images, and then to subtract the registered second image from the
Discussion
Numerous studies have shown that skeletal development and skeletal regeneration after surgery or injury are dependent on the formation of vascular tissues [5], [19], [24], [25]. Given that dysmorphic development of vascular tissues is related to a multitude of pathologies, a central focus of much of vascular tissue research is directed at the mechanisms associated with vascular morphogenesis [26], [27]. While previous studies on re-vascularization of bone injuries have focused primarily on
Acknowledgments
Supported with grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (PO1AR049920 (TAE and LCG) and AR052746 and S10 RR021072 (EFM)). Institutional support was provided by the Department of Orthopaedic Surgery Boston University School of Medicine and by Boston University School of Medicine.
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Supported with grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (PO1AR049920 (TAE and LCG) and S10 RR021072 (EFM)). Institutional support was provided by the Department of Orthopaedic Surgery Boston University School of Medicine and by Boston University School of Medicine.