The worldwide incidence of bone disorders and conditions has trended steeply upward and is expected to double by 2020, especially in populations where aging is coupled with increased obesity and poor physical activity. Engineered bone tissue has been viewed as a potential alternative to the conventional use of bone grafts, due to their limitless supply and no disease transmission. However, bone tissue engineering practices have not proceeded to clinical practice due to several limitations or challenges. Bone tissue engineering aims to induce new functional bone regeneration via the synergistic combination of biomaterials, cells, and factor therapy. In this review, we discuss the fundamentals of bone tissue engineering, highlighting the current state of this field. Further, we review the recent advances of biomaterial and cell-based research, as well as approaches used to enhance bone regeneration. Specifically, we discuss widely investigated biomaterial scaffolds, micro- and nano-structural properties of these scaffolds, and the incorporation of biomimetic properties and/or growth factors. In addition, we examine various cellular approaches, including the use of mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), and platelet-rich plasma (PRP), and their clinical application strengths and limitations. We conclude by overviewing the challenges that face the bone tissue engineering field, such as the lack of sufficient vascularization at the defect site, and the research aimed at functional bone tissue engineering. These challenges will drive future research in the field.

Skeletal muscle size is regulated by anabolic (hypertrophic) and catabolic (atrophic) processes. We first characterized molecular markers of both hypertrophy and atrophy and identified a small subset of genes that are inversely regulated in these two settings (e.g. upregulated by an inducer of hypertrophy, insulin-like growth factor-1 (IGF-1), and down-regulated by a mediator of atrophy, dexamethasone). The genes identified as being inversely regulated by atrophy, as opposed to hypertrophy, include the E3 ubiquitin ligase MAFbx (also known as atrogin-1). We next sought to investigate the mechanism by which IGF-1 inversely regulates these markers, and found that the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/ mTOR) pathway, which we had previously characterized as being critical for hypertrophy, is also required to be active in order for IGF-1-mediated transcriptional changes to occur. We had recently demonstrated that the IGF1/PI3K/Akt pathway can block dexamethasoneinduced up-regulation of the atrophy-induced ubiquitin ligases MuRF1 and MAFbx by blocking nuclear translocation of a FOXO transcription factor. In the current study we demonstrate that an additional step of IGF1 transcriptional regulation occurs downstream of mTOR, which is independent of FOXO. Thus both the Akt/FOXO and the Akt/mTOR pathways are required for the transcriptional changes induced by IGF-1.Skeletal muscle mass and fiber size is regulated in response to changes in workload, activity, conditions such as AIDS, cancer, and aging, and by cachectic glucocorticoids such as dexamethasone (1-3). An increase in adult muscle mass and fiber size is called "hypertrophy" and is associated with increased protein synthesis (4). A decrease in mass, called "atrophy," is characterized by enhanced protein degradation (3,5,6).Hypertrophy in adult skeletal muscle is accompanied by the increased expression of insulin-like growth factor-1 (IGF-1) 1 (4, 7). When IGF-1 was overexpressed in the skeletal muscle of transgenic mice an increase in muscle size resulted (8, 9). Furthermore, addition of IGF-1 in vitro to differentiated muscle cells promotes myotube hypertrophy (10 -12), supporting the idea that IGF-1 is sufficient to induce hypertrophy. The binding of IGF-1 to its receptor triggers the activation of phosphatidylinositol 3-kinase (PI3K). PI3K phosphorylates the membrane phospholipid phosphatidylinositol 4,5-bisphosphate to produce phosphatidylinositol 3,4,5-trisphosphate (13, 14), creating a lipid binding site on the cell membrane for the serine/threonine kinase Akt (also called Akt1 and PKB, for protein kinase B) (15-17). The subsequent translocation of Akt to the membrane facilitates its phosphorylation and activation by the kinase PDK-1 (14, 18, 19). Cell growth and survival in a variety of tissues and cell types in response to IGF-1, insulin, and other growth factors is critically mediated by Akt (14, 19). Direct and indirect targets downstream of Akt include the mammalian target of rapamycin (mTOR), p70S6K, and PHAS-1...

Large-area or critical-sized bone defects pose a serious challenge in orthopedic surgery, as all current treatment options present with shortcomings. Bone tissue engineering offers a more promising alternative treatment strategy. However, this approach requires mechanically stable scaffolds that support homogenous bone formation throughout the scaffold thickness. Despite advances in scaffold fabrication, current scaffold-based techniques are unable to support uniform, three-dimensional bone regeneration, and are limited to only the scaffold surface in vitro and in vivo. This is mainly because of inadequate scaffold pore sizes (<200 μm) and accessible pore volume, and the associated limited oxygen diffusion and vascular invasion. In this study, we have adopted a method combining microsphere-sintering and porogen-leaching techniques to fabricate scaffolds with an increased accessible pore volume. Of the scaffolds developed, moderately porous poly(85 lactide-co-15 glycolide) (PLGA) microsphere scaffolds were selected as most advantageous, since they retain mechanical strength in the range of human cancellous bone and display a significantly higher accessible pore volume, which is attributed to an increased percentage of larger pores (i.e., size range 200-600 μm). Unlike control scaffolds with a limited pore size and an accessible pore volume, moderately porous scaffolds displayed increased oxygen diffusion, pre-osteoblast cell infiltration, proliferation, and survival throughout the entire scaffold. Furthermore, moderately porous PLGA microsphere scaffolds displayed enhanced and homogenous mineralization in vitro. Since these newly designed moderately porous scaffolds are weight bearing, are fully osteoconductive, and have the ability to support vascularization, they may serve as effective scaffolds for large-area bone defect repair/regeneration. In addition, this study demonstrates the ability to modulate scaffold porosity and, in turn, to develop oxygen tension-controlled matrices that are effective for large-area bone regeneration.

Presently, orthopedic and oral/maxillofacial implants represent a combined $2.8 billion market, a figure expected to experience significant and continued growth. Although traditional permanent implants have been proved clinically efficacious, they are also associated with several drawbacks, including secondary revision and removal surgeries. Non-permanent, biodegradable implants offer a promising alternative for patients, as they provide temporary support and degrade at a rate matching tissue formation, and thus, eliminate the need for secondary surgeries. These implants have been in clinical use for nearly 25 years, competing directly with, or maybe even exceeding, the performance of permanent implants. The initial implantation of biodegradable materials, as with permanent materials, mounts an acute host inflammatory response. Over time, the implant degradation profile and possible degradation product toxicity mediate long-term biodegradable implant-induced inflammation. However, unlike permanent implants, this inflammation is likely to cease once the material disappears. Implant-mediated inflammation is a critical determinant for implant success. Thus, for the development of a proactive biodegradable implant that has the ability to promote optimal bone regeneration and minimal detrimental inflammation, a thorough understanding of short- and long-term inflammatory events is required. Here, we discuss an array of biodegradable orthopedic implants, their associated short- and long- term inflammatory effects, and methods to mediate these inflammatory events.

For tissue engineering applications, effective bone regeneration requires rapid neo-vascularization of implanted grafts to ensure the survival of cells in the early post-implantation phase. Incorporation of autologous endothelial progenitor cells (EPCs) for the promotion of primitive vascular network formation ex vivo has offered great promise for improved graft survival, enhanced rate of vascularization and bone regeneration in vivo. For clinical usage, identification of an optimal EPC isolation source from the patient is critical. We have, for the first time, characterized and directly compared EPCs from rabbit peripheral blood and bone marrow (PB-EPCs and BM-EPCs, respectively). PB-EPCs outperformed BM-EPCs on all measures. PB-EPCs displayed typical endothelial cell markers, such as CD31, as well as high angiogenic potential in three-dimensional extracellular matrix in vitro. Furthermore, PB-EPCs cultured simultaneously with mesenchymal stem cells, displayed significantly enhanced expression levels of key osteogenic and vascular markers, including alkaline phosphatase, bone morphogenetic protein 2, and vascular endothelial growth factor. On the contrary, putative BM-EPCs did not express CD31, and instead, expressed key smooth muscle markers. BM-EPCs further failed to display vasculogenic activity. Hence, the highly angiogenic PB-derived EPCs may serve as an ideal cell population for enhanced vascularization and success of engineered bone tissue. ß

Designing biodegradable scaffolds with bone-compatible mechanical properties has been a significant challenge in the field of bone tissue engineering and regenerative engineering. The objective of this work is to improve the polymeric scaffold's mechanical strength by compositing it with mechanically superior carbon nanotubes. Poly(lactide-co-glycolide) (PLGA) microsphere scaffolds exhibit mechanical properties in the range of human cancellous bone. On the other hand, carbon nanotubes have outstanding mechanical properties. The aim of this study is to improve further the mechanical strength of PLGA scaffolds such that they may be applicable for a wide range of load-bearing repair and regeneration applications. We have formed composite microspheres of PLGA containing pristine and modified (with hydroxyl (OH), carboxylic acid (COOH)) multi-walled carbon nanotubes (MWCNTs), and fabricated them into three-dimensional porous scaffolds. Results show that by adding only 3% MWCNTs, the compressive strength and modulus was significantly increased (35 MPa, 510.99 MPa) compared to pure PLGA scaffolds (19 MPa and 166.38 MPa). Scanning electron microscopy images showed excellent cell adhesion and proliferation. In vitro studies exhibited good cell viability, proliferation and mineralization. The in vivo study, however, indicated differences in inflammatory response throughout the 12 weeks of implantation, with OH-modified MWCNTs having the least response, followed by unmodified and COOH-modified exhibiting a more pronounced response. Overall, our results show that PLGA scaffolds containing water-dispersible MWCNTs are mechanically stronger and display good cellular and tissue compatibility, and hence are potential candidates for load-bearing bone tissue engineering.

Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. Besides its role in normal physiology, angiogenesis is significantly involved in many pathological conditions, including inflammation, cardiovascular diseases and cancer. Numerous studies have been undertaken in the area of tumor angiogenesis. It is known that pathological angiogenesis is necessary for tumors to proceed from avascular, dormant stage to vascular, sprouting stage and also contributes to their later invasion and metastasis. Playing a central role in tumor angiogenesis, vascular endothelial growth factor is considered as a key target in therapeutic approaches. This article aims to review the critical role of VEGF in tumor angiogenesis and the importance of VEGF-targeted strategies in cancer treatment.

The success of a clinically-applicable bone tissue engineering construct for large area bone defects depends on its ability to allow for homogeneous bone regeneration throughout the construct. Insufficient vascularization, and consequently inadequate oxygen tension, throughout constructs has been largely cited as the most significant obstacle facing successful bone regeneration in large area defects. The development of constructs that support bone and vessel-forming cell growth and function throughout the scaffold structure are desired for large-area bone defect repair. Here, we developed oxygen tension-controlled matrices that support more homogenous oxygen levels throughout the constructs. Specifically, we examined polylactic co-glycolic acid (PLGA) scaffolds with optimized pore distribution and the percent pore volumes, and demonstrated significantly decreased oxygen and pH gradient from the exterior of the construct to the interior after long-term cell culture in vitro. We confirmed the ability of these optimized constructs to support the cellular survival via live/dead assay. In addition, we examined their ability to support the maintenance of two clinically relevant progenitor cell populations for bone tissue engineering and vascularization, namely mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs), and confirmed the expression of key bone and vascular markers via immunofluorescence.