Current Pharmaceutical Design - Volume 19, Issue 19, 2013

There remains a great need to develop therapeutic agents to treat critical size defects and non-union fractures and one of the potential agents is recombinant human fibroblast growth factor 2 (FGF-2). We discuss the function of FGF-2 in bone formation, bone resorption, and downstream signaling pathways and review the role of exogenous FGF-2 in fracture healing. The importance of endogenous FGF-2 in bone formation and its potential importance in fracture healing in response to parathyroid hormone (PTH) and bone morphogenetic protein 2 (BMP2) is described. In addition we will review, FGF-2 signaling crosstalk with Wnt signaling and PTH signaling in bone formation and repair. Finally, we discuss the outstanding unresolved issues in the application of FGF-2 as therapeutic agent for bone regeneration.

Growth differentiation factor 5 (GDF5) is a member of the bone morphogenic protein (BMP) family and plays critical roles in organ development processes including bone, cartilage, ligament, and joint formation. GDF5 is expressed in the cartilage primordium in the early limb development, and in the interzone of joint formation sites. GDF5 is also observed in adult tissue and cell lines. This spatialtemporal expression pattern of GDF5 proves its essential role in the formation of bone and cartilage. Similar to other members of BMPs, the signaling cascade of GDF5 is originated through binding to type I and type II receptors and thus regulating the downstream intracellular biochemical processes. Mutations of GDF5 are associated with several human and animal diseases that are characterized by skeletal deformity such as short digits and short limbs. In vitro and in vivo studies demonstrated that overexpression of GDF5 or administration of recombinant protein promotes chondrogenesis and osteogenesis. Moreover, a promising feature of GDF5 is osteoinduction, which is used in tissue engineering for bone repair with or without a carrier in animal platforms and in human preclinical settings. The exciting results signify that GDF5 is a compelling candidate for bone tissue engineering by enhancing osteogenesis and angiogenesis. In this review, we will focus the discussion on the basic structure, signaling pathways, function in cartilage and bone formation, and potential clinical application of GDF5 in bone tissue regeneration.

Both osteogenesis and angiogenesis are integrated parts of bone growth and regeneration. Combined delivery of osteogenic and angiogenic factors is a novel approach in bone regenerative engineering. Exogenous addition of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) together with an osteoconductive scaffold is a very promising method to enhance bone repair. This concept has been incorporated into the development of new strategies for bone tissue engineering and significant advancements have been made in last 10 years. In contrary to previous belief that VEGF modulates bone repair only by enhancing angiogenesis in the proximity of bone injury, recent evidence also suggests that cross-talk between VEGF and BMP signaling pathways in MSCs promotes osteoblastic differentiation of MSCs which aids in fracture repair. Future studies should focus on cross-talk between angiogenesis and osteogenesis, optimization of VEGF-BMP ratios, selection of the most potent BMPs, and optimization of delivery methods for VEGF and BMP. Recent discoveries from basic research including effective delivery of growth factors and cells to the area of interest will help bring VEGF plus BMP for bone healing from the bench to the patient’s bedside.

Recombinant human PDGF BB homodimer (rhPDGF-BB) is a potent recruiter of, and strong mitogenic factor for, cells crucial to musculoskeletal tissue repair, including mesenchymal stem cells (MSCs), osteogenic cells and tenocytes. rhPDGF-BB also upregulates angiogenesis. These properties allow rhPDGF-BB to trigger the cascade of bone and adjoining soft tissue repair and regeneration. This mechanism of action has been established in numerous preclinical and clinical studies. Demonstration of the safety and efficacy of rhPDGF-BB in the healing of chronic foot ulcers in diabetic patients and regeneration of alveolar (jaw) bone lost due to chronic infection from periodontal disease has resulted in two FDA-approved products based on this molecule. A third product is in late stages of clinical development, with pilot and pivotal clinical studies of rhPDGF-BB mixed with an osteoconductive bone matrix (Augment® Bone Graft) in foot and ankle fusions demonstrating that this product is at least as effective as bone autograft, and has an improved safety profile. Additional combinations of rhPDGF-BB with tissue-specific matrices are also being studied clinically in additional musculoskeletal indications.

Due to their short half-life and diffusion away from the site of regeneration, osteogenic proteins, used in regenerative medicine, require high doses leading to undesirable side effects. An exciting approach is to utilize peptides derived from the active domains of soluble and insoluble components of the bone extracellular matrix (ECM) to initiate the cascade of osteogenesis, vasculogenesis, mineralization, and bone formation. Osteogenic peptides derived from bone morphogenetic proteins, integrin-binding and heparin-binding peptides, collagen-derived peptides, peptides derived from bone sialoprotein and enamel matrix proteins, vasoconstrictive peptides, peptides derived from the histone proteins involved in the structure of chromatin and those derived from receptor-binding domain of human thrombin, peptides that abolish the expression of tumor necrosis factor-α, and neuropeptides have been discovered. In this work, anabolic effects of osteogenic peptides in particular with respect to cell adhesion, differentiation, and mineralization are discussed. These peptides show significantly higher biological activity in vitro and in vivo, in small animal models, when immobilized in a matrix by conjugation or grafting, most likely due to the peptide mobility and its diffusion away from the site of regeneration. Combination of osteogenic and integrin- binding peptides synergistically enhances cell adhesion and mineralization. Functionalization of orthopedic implants with osteogenic peptides can improve cell adhesion, mineralization, and overall integration of the implant with the surrounding tissue, which in turn reduces implant loosening and failure. The use of osteogenic peptides as an alternative growth factor in orthopedic applications rests on future in vivo studies that evaluate these peptides in primates and humans.

Regeneration of bone requires the coordinated processes of angiogenesis and osteogenesis. These repair mechanisms are closely linked through both direct cell-cell contact and indirect paracrine signaling among osteoblasts, endothelial cells, and other cell types. The vasculature provides a source of nutrients, oxygen, metabolic substrates, and access for circulating cells that help to support new bone formation. The complexity of the endogenous signaling axis that promotes angiogenesis provides numerous opportunities for therapeutic intervention ranging from progenitor cell mobilization to endothelial proliferation and sprouting. Small molecules are particularly appealing for regenerative medicine applications because many exhibit extended in vivo stability, low cost, and scalable production. Innovative techniques for developing small molecules such as high throughput functional assays and broad-spectrum database analysis techniques have led to the development of new compounds and the identification of novel applications of existing drugs. In addition, rapid advances in biomaterials design and synthesis provide platforms to deliver therapeutic small molecules to sites of bone injury. This review presents an overview of current strategies for harnessing endogenous healing mechanisms using small molecules by targeting angiogenesis, osteogenesis, or both.

Tissue engineering aims to repair, restore, and regenerate lost or damaged tissues by using biomaterials, cells, mechanical forces and factors (chemical and biological) alone or in combination. Growth factors are routinely used in the tissue engineering approach to expedite the process of regeneration. The growth factor approach has been hampered by several complications including high dose requirements, lower half-life, protein instability, higher costs and undesired side effects. Recently a variety of alternative small molecules of both natural and synthetic origin have been explored as alternatives to growth factors for tissue regeneration applications. Small molecules are simple biochemical components that elicit certain cellular responses through signaling cascades. Small molecules present a viable alternative to biological factors. Small molecule strategies can reduce various side effects, maintain bioactivity in a biological environment and minimize cost issues associated with complex biological growth factors. This manuscript focuses on three-osteoinductive small molecules, namely melatonin, resveratrol (from natural sources) and purmorphamine (synthetically designed) as inducers of bone formation and osteogenic differentiation of stem cells. Efforts have been made to summarize possible biological pathways involved in the action of each of these drugs. Melatonin is known to affect Mitogen Activated Protein (MAP) kinase, Bone morphogenic protein (BMP) and canonical wnt signaling. Resveratrol is known to activate cascades involving Wnt and NAD-dependent deacetylase sirtuin-1 (Sirt1). Purmorphamine is a Hedgehog (Hh) pathway agonist as it acts on Smoothened (Smo) receptors. These mechanisms and the way they are affected by the respective small molecules will also be discussed in the manuscript.

The field of regenerative medicine and tissue engineering is an ever evolving field that holds promise in treating numerous musculoskeletal diseases and injuries. An important impetus in the development of the field was the discovery and implementation of stem cells. The utilization of mesenchymal stem cells, and later embryonic and induced pluripotent stem cells, opens new arenas for tissue engineering and presents the potential of developing stem cell-based therapies for disease treatment. Multipotent and pluripotent stem cells can produce various lineage tissues, and allow for derivation of a tissue that may be comprised of multiple cell types. As the field grows, the combination of biomaterial scaffolds and bioreactors provides methods to create an environment for stem cells that better represent their microenvironment for new tissue formation. As technologies for the fabrication of biomaterial scaffolds advance, the ability of scaffolds to modulate stem cell behavior advances as well. The composition of scaffolds could be of natural or synthetic materials and could be tailored to enhance cell self-renewal and-or direct cell fates. In addition to biomaterial scaffolds, studies of tissue development and cellular microenvironments have determined other factors, such as growth factors and oxygen tension, that are crucial to the regulation of stem cell activity. The overarching goal of stem cell-based tissue engineering research is to precisely control differentiation of stem cells in culture. In this article, we review current developments in tissue engineering, focusing on several stem cell sources, induction factors including growth factors, oxygen tension, biomaterials, and mechanical stimulation, and the internal and external regulatory mechanisms that govern proliferation and differentiation.

Worldwide, more than 2.2 million patients undergo bone graft procedures annually. In each of these procedures an interface is formed between the host tissue and the graft material. Synthetic implants exhibit an interface with the host tissue and the formation of a homogenous interface consisting of bone and void of intervening soft tissue is desired (osseointegration); recent developments have highlighted the benefit of incorporating nanostructures at that interface. Autograft and allograft bone are frequently used for bone loss, nonunion fractures, and spinal fusions; however, both are plagued with complications either due to supply or inadequate graft properties. In contrast to bone tissue engineering, which uses a top-down approach to repair bone defects, bone regenerative engineering uses a bottomup approach focused on strategies incorporating stem cells, biomaterials, and growth factors alone or in combination to generate or regenerate bone tissue. Early constructs developed for bone regenerative engineering utilized polymeric microstructures, presenting surface features with characteristic dimensions similar to that of a cell (1μm – 1000μm). These microstructures were typically biodegradable and demonstrated an excellent ability to match the mechanics of native bone tissue. They were also osteoconductive—capable of promoting osteoblast growth. On the other hand, the osteoinductive abilities of these microstructures were lacking. Osteoinduction, or the ability to promote the progression of a preosteoblastic cell to a mature osteoblast, historically was achieved in two ways: via the addition of nanoscale ceramics to the microstructures or via an external stimulus such as the addition of bone morphogenetic proteins (BMPs). More recent developments in bone regenerative engineering have utilized polymeric nanostructures (less than 1μm) with characteristic dimensions an order of magnitude or more less than that of a cell to stimulate and drive an osteoinductive response in the absence of growth factors. Despite strong literature evidence supporting the nanostructures’ ability to be both osteoconductive and osteoinductive, there is still disparity regarding how nanostructures regulate the progression towards an osteoblastic phenotype. This review will explore unique micro- and nano-architectures, how they initiate osteoinductive signals through pathways similar to BMPs, and how these unique geometries can be translated to the clinic.

There is a profound need for orthopaedic grafting strategies due to various trauma and musculoskeletal diseases. Tissue engineering offers a promising avenue to develop viable grafts for bone repair. The transfer of bone tissue engineering strategies to clinical applications is limited by the failure to adequately vascularize scaffolds after implantation. This review focuses on the natural processes for bone and vessel formation as well as the microenvironmental cues and microscale fabrication techniques to properly coordinate these events towards successful vascularization of tissue engineered scaffolds.

Nonunions and delayed unions are among the more challenging clinical and surgical entities an orthopaedic surgeon must manage. Effective strategies that address these complex problems are in need and gene therapy represents a potential therapeutic option. Among the many properties that bone morphogenetic proteins (BMPs) possess, their potent osteoinductive effects make them attractive growth factors for use in gene therapy to address large bony defects. Gene therapy enables a sustained production of BMP to be achieved at specific sites of interest and represents a significant advantage over protein-delivery based systems. Viruses are effective vectors for delivering BMP cDNA because they are designed to efficiently infect cells and transmit genetic material. However, safety concerns such as immune system activation and insertional mutagenesis represent drawbacks that may limit their clinical efficacy. Nonviral vectors are emerging as attractive candidates for gene delivery since they avoid many of the safety issues seen with viral vectors but have lower genetic transfer efficiency. A wide variety or preclinical studies of bone regeneration using BMPs have demonstrated the efficacy of both in vivo and ex vivo gene therapy techniques and these will be explored in this review article.

Cystic fibrosis (CF) is a common inherited fatal disease affecting 70,000 people worldwide, with a median predicted age of survival of approximately 38 years. The deletion of Phenylalanine in position 508 of the Cystic Fibrosis Transmembrane conductance Regulator (F508del-CFTR) is the most common mutation in CF patients: the deleted protein, not properly folded, is degraded. To date no commercial drugs are available. Low temperature, some osmolytes and conditions able to induce heat shock protein 70 (Hsp70) expression and heat shock cognate 70 (Hsc70) inhibition result in F508del-CFTR rescue, hence restoring its physiological function: this review sheds light on the correlation between these several evidences. Interestingly, all these approaches have a role in the cell stress response (CSR), a set of cell reactions to stress. In addition, unpredictably, F508del-CFTR rescue has to be considered in the frame of CSR: entities that induce - or are induced during - the CSR are, in general, also able to correct trafficking defect of CFTR. Specifically, the low temperature induces, by definition, a CSR; osmolytes, such as glycerol and trimethylamine N-oxide (TMAO), are products of the CSR; pharmacological correctors, such as Matrine and 4-phenylbutirric acid (4PBA), down-regulate the constitutive Hsc70 in favor of an up-regulation of the inducible chaperone Hsp70, another component of the CSR. The identification of a common mechanism of action for different types of correctors could drive the discovery of new active molecules in CF, overcoming methods clinically inapplicable, such as the low temperature.

Correcting multiple defects of mutant CFTR with small molecule compounds has been the goal of an increasing number of recent Cystic Fibrosis (CF) drug discovery programmes. However, the mechanism of action (MoA) by which these molecules restore mutant CFTR is still poorly understood, in particular of CFTR correctors, i.e., compounds rescuing to the cells surface the most prevalent mutant in CF patients - F508del-CFTR. However, there is increasing evidence that to fully restore the multiple defects associated with F508del-CFTR, different small molecules with distinct corrective properties may be required. Towards this goal, a better insight into MoA of correctors is needed and several constraints should be addressed. The methodological approaches to achieve this include: 1) testing the combined effect of compounds with that of other (non-pharmacological) rescuing strategies (e.g., revertants or low temperature); 2) assessing effects in multiple cellular models (non-epithelial vs epithelial, non-human vs human, immortalized vs primary cultures, polarized vs non polarized, cells vs tissues); 3) assessing compound effects on isolated CFTR domains (e.g., compound binding by surface plasmon resonance, assessing effects on domain folding and aggregation); and finally 4) assessing compounds specificity in rescuing different CFTR mutants and other mutant proteins. These topics are reviewed and discussed here so as to provide a state-of-the art review on how to combine multiple ways of rescuing mutant CFTR to the ultimate benefit of CF patients.

Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene that encodes the CF transmembrane conductance regulatory (CFTR) protein. The majority of wild-type CFTR and virtually all mutant ΔF508 CFTR are degraded before reaching the cell surface. Certain agents and conditions that increase expression and maturation of CFTR enable the protein to function at the cell surface. We and several research groups have reported that S-nitrosoglutathione (GSNO), a class of endogenous S-nitrosothiols, increases the maturation and function of CFTR in human airway epithelial cells. S-nitrosothiols (SNOs) are endogenous molecules with several cell signaling effects and potential relevance to human lung disease. SNOs are normally present in the human airway and have beneficial effects on lung function. Biochemical evidence suggests that SNOs act on post-translational protein modifications through mechanisms involving S-nitrosylation reactions. S-nitrosylation reactions are increasingly recognized to represent metabolically regulated cell signaling processes. Airway epithelial S-nitrosylation signaling disorders have been observed in a range of diseases, including CF. SNO levels are low in CF patients and normal physiological concentrations are effective in increasing CFTR maturation. The mechanisms by which SNOs improve CFTR expression appear to be novel. However, the precise mechanisms by which SNOs exert their beneficial effects are poorly understood. In the near future, we expect to identify the novel mechanisms by which SNO augments CFTR maturation. This information will be critical for optimizing the design and dosing of SNOs that might be used as CFTR corrector therapies in clinical trials.

Genistein and curcumin are major components of Asian foods, soybean and curry turmeric respectively. These compounds have been intensively investigated for their chemical and biological features conferring their anti-cancer activity. Genistein and curcumin have also been investigated for their potentiation effects on disease-associated CFTR mutants such as ΔF508 and G551D. Recently, we investigated the combined effect of genistein and curcumin on G551D-CFTR, which exhibits gating defects without abnormalities in protein synthesis or trafficking using the patch-clamp technique. We found that genistein and curcumin showed additive effects on their potentiation of G551D-CFTR in high concentration range and also, more importantly, showed a significant synergistic effect in their minimum concentration ranges. These results are consistent with the idea that multiple mechanisms are involved in the action of these CFTR potentiators. In this review, we revisit the pharmacology of genistein and curcumin on CFTR and also propose new pharmaceutical implications of combined use of these compounds in the development of drugs for CF pharmacotherapy.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated Cl- channel whose major function is to facilitate epithelial fluid secretion. Loss-of-function mutations in CFTR cause the genetic disease cystic fibrosis. CFTR is required for transepithelial fluid transport in certain secretory diarrheas, such as cholera, and for cyst expansion in autosomal dominant polycystic kidney disease. High-throughput screening has yielded CFTR inhibitors of the thiazolidinone, glycine hydrazide and quinoxalinedione chemical classes. The glycine hydrazides target the extracellular CFTR pore, whereas the thiazolidinones and quinoxalinediones act at the cytoplasmic surface. These inhibitors have been widely used in cystic fibrosis research to study CFTR function at the cell and organ levels. The most potent CFTR inhibitor has IC50 of approximately 4 nM. Studies in animal models support the development of CFTR inhibitors for antisecretory therapy of enterotoxin-mediated diarrheas and polycystic kidney disease.