Oxidized alginate hydrogels with the GHK peptide enhance cord blood mesenchymal stem cell osteogenesis: A paradigm for metabolomics-based evaluation of biomaterial design.

Oxidized alginate hydrogels are appealing alternatives to natural alginate due to their favourable biodegradability profiles and capacity to self-crosslink with amine containing molecules facilitating functionalization with extracellular matrix cues, which enable modulation of stem cell fate, achieve highly viable 3-D cultures, and promote cell growth. Stem cell metabolism is at the core of cellular fate (proliferation, differentiation, death) and metabolomics provides global metabolic signatures representative of cellular status, being able to accurately identify the quality of stem cell differentiation. Herein, umbilical cord blood mesenchymal stem cells (UCB MSCs) were encapsulated in novel oxidized alginate hydrogels functionalized with the glycine-histidine-lysine (GHK) peptide and differentiated towards the osteoblastic lineage. The ADA-GHK hydrogels significantly improved osteogenic differentiation compared to gelatin-containing control hydrogels, as demonstrated by gene expression, alkaline phosphatase activity and bone extracellular matrix deposition. Metabolomics revealed the high degree of metabolic heterogeneity in the gelatin-containing control hydrogels, captured the enhanced osteogenic differentiation in the ADA-GHK hydrogels, confirmed the similar metabolism between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. Our results suggest a novel paradigm for metabolomics-guided biomaterial design and robust stem cell bioprocessing. STATEMENT OF SIGNIFICANCE: Producing high quality engineered bone grafts is important for the treatment of critical sized bone defects. Robust and sensitive techniques are required for quality assessment of tissue-engineered constructs, which result to the selection of optimal biomaterials for bone graft development. Herein, we present a new use of metabolomics signatures in guiding the development of novel oxidised alginate-based hydrogels with umbilical cord blood mesenchymal stem cells and the glycine-histidine-lysine peptide, demonstrating that GHK induces stem cell osteogenic differentiation. Metabolomics signatures captured the enhanced osteogenesis in GHK hydrogels, confirmed the metabolic similarity between differentiated cells and primary osteoblasts, and elucidated the metabolic mechanism responsible for the function of GHK. In conclusion, our results suggest a new paradigm of metabolomics-driven design of biomaterials.

Metabolomics Analysis of the Osteogenic Differentiation of Umbilical Cord Blood Mesenchymal Stem Cells Reveals Differential Sensitivity to Osteogenic Agents.

Mesenchymal stem cells (MSCs) of fetal origin, such as umbilical cord blood MSCs (UCB MSCs), have emerged as a promising cell source for musculoskeletal tissue regeneration because of their higher proliferation potential, lack of donor site morbidity, and their off-the-shelf potential. MSCs differentiated toward the osteogenic lineage exhibit a specific metabolic phenotype characterized by reliance to oxidative phosphorylation for energy production and reduced glycolytic rates. Currently, limited information exists on the metabolic transitions at different stages of the osteogenic process after osteoinduction with different agents. Herein, the osteoinduction efficiency of BMP-2 and dexamethasone on UCB MSCs was assessed using gas chromatography-mass spectrometry (GC-MS) metabolomics analysis, revealing metabolic discrepancies at 7, 14, and 21 days of induction. Whereas both agents when administered individually were able to induce collagen I, osteocalcin, and osteonectin expression, BMP-2 was less effective than dexamethasone in promoting alkaline phosphatase expression. The metabolomics analysis revealed that each agent induced distinct metabolic alterations, including changes in amino acid pools, glutaminolysis, one-carbon metabolism, glycolysis, and tricarboxylic acid cycle. Importantly, we showed that in vitro-differentiated UCB MSCs acquire a metabolic physiology similar to primary osteoblasts when induced with dexamethasone but not with BMP-2, highlighting the fact that metabolomics analysis is sensitive enough to reveal potential differences in the osteogenic efficiency and can be used as a quality control assay for evaluating the osteogenic process.

Fibronectin stimulates the osteogenic differentiation of murine embryonic stem cells.

Conditioned medium from human hepatocarcinoma cells (HepG2-CM) has been shown to stimulate the osteogenic/chondrogenic differentiation of murine embryonic stem cells (mESCs). HepG2-CM is considered to contain visceral endoderm (VE)-like signals and attempts have recently been made to characterize it, using proteomic profiling, with fibronectin being identified as one promising candidate. Herein, we investigated whether fibronectin is able to mimic the activities of HepG2-CM during the osteogenic differentiation of mESCs. Specifically, the addition of RGD peptides and heparin in HepG2-CM significantly reduced the growth- and adhesion-promoting effects of HepG2-CM, in addition to suppressing its osteogenic-inductive activity. Furthermore, direct addition of fibronectin to basal medium was able to reproduce, at least partially, the function of HepG2-CM. In particular, fibronectin induced the early onset of osteogenic differentiation in mESCs, as confirmed by gene expression of osteogenic markers, and resulted in the three-fold higher calcium deposition at day 11 of osteogenic culture compared to the control group. These data clearly suggest that fibronectin contributes to the biological activities of HepG2-CM and plays a stimulatory role during the process of osteogenesis in mESCs. Copyright © 2015 John Wiley & Sons, Ltd.

Investigational drugs for fracture healing: preclinical & clinical data.

INTRODUCTION: The need for fracture healing enhancement for the management of fracture complications such as non-union and for the achievement of early function in fracture patients is constantly increasing. Therefore, the development and evaluation of novel pharmaceutical agents is mandatory in order to accelerate the process and increase bone union rates. AREAS COVERED: This review summarizes the most recent knowledge on the pharmacological enhancement of fracture repair. It provides a synopsis of the most important preclinical and clinical studies published over the past five years on long bone fracture healing. EXPERT OPINION: To date, limited drugs seem to have the potential for clinical use in fracture healing enhancement and the field is progressing very slowly. Among anti-osteoporotic drugs, only PTH and anti-sclerostin antibodies have such a potential but further research is needed before clinical use. The same applies also to BMPs, the use of which still carries major drawbacks that should be overcome before their widespread clinical utilization. Other drugs and growth factors, such as statins, VEGF, FGF, EPO, could be future key players in fracture healing but evidence is still lacking. Further in depth understanding of the healing process is essential in order to identify novel effective pharmacological agents.

Successful long-term mandibular reconstruction and rehabilitation using non-vascularised autologous bone graft and recombinant human BMP-7 with subsequent endosseous implant in a patient with bisphosphonate-related osteonecrosis of the jaw.

We describe a case of extensive osteonecrosis of the mandible after a dental extraction in a 71-year-old woman who was taking alendronic acid (Fosamax(®), Merck) for osteoporosis. Bone damaged by bisphosphonate-related osteonecrosis of the jaw (BRONJ), also now known as medication-related osteonecrosis of the jaw (MRONJ), can be regenerated and filled with endosseous implants using non-vascularised autologous grafts.

Bone and cartilage regeneration with the use of umbilical cord mesenchymal stem cells.

INTRODUCTION: The production of functional alternatives to bone autografts and the development new treatment strategies for cartilage defects are great challenges that could be addressed by the field of tissue engineering. Umbilical cord mesenchymal stem cells (MSCs) can be used to produce cost-effective, atraumatic and possibly autologous bone and cartilage grafts. AREAS COVERED: MSCs can be isolated from umbilical cord Wharton's jelly, perivascular tissue and blood using various techniques. Those cells have been characterized and phenotypic similarities with bone marrow-derived MSCs (BM-MSCs) and embryonic stem cells have been found. Findings on their differentiation into the osteogenic and chondrogenic lineage differ between studies and are not as consistent as for BM-MSCs. EXPERT OPINION: MSCs from umbilical cords have to be more extensively studied and the mechanisms underlying their differentiation have to be clarified. To date, they seem to be an attractive alternative to BM-MSCs. However, further research with suitable scaffolds and growth factors as well as with novel scaffold fabrication and culture technology should be conducted before they are introduced to clinical practice and replace BM-MSCs.

Ludwig's Angina: The Original Angina.

Ludwig's angina was first detailed by the German surgeon Wilhelm Friedrich von Ludwig in 1836. We present a case which needed awake fibreoptic intubation due to severe trismus and a prolonged period intubated in the Intensive Care Unit after incision and drainage of neck spaces and removal of his lower wisdom teeth. He was finally discharged a week after admission and followed up in the outpatient clinic. The case is presented with clinical photographs and a video of the fibreoptic intubation to illustrate the airway.

Anti-inflammatory role and immunomodulation of mesenchymal stem cells in systemic joint diseases: potential for treatment.

INTRODUCTION: Mesenchymal stem cells (MSCs) are multipotent stromal cells characterized by their ability to differentiate into adipocytes, chondrocytes, osteocytes and a number of other lineages. Investigation into their use has increased in recent years as characterization of their immunomodulatory properties has developed, and their role in the pathophysiology of joint disease has been suggested. AREAS COVERED: MSCs demonstrate immunosuppressive functionality by suppressing T- and B-cell responses following activation by cytokines such as IL-6 and IL-1α. They also can be induced to exert pro-inflammatory effects in the presence of acute inflammatory environment due to the actions of TNF-α and IFN-γ. In inflammatory joint diseases such as rheumatoid arthritis, MSCs in bone marrow migrate to joints by a TNF-α-dependent mechanism and may be in part responsible for the disease process. MSCs have also been demonstrated in increased numbers in periarticular tissues in osteoarthritis, which may reflect an attempt at joint regeneration. EXPERT OPINION: Clinical applications for MSCs have shown promise in a number of inflammatory and autoimmune disorders. Future work is likely to further reveal the immunosuppressive characteristics of MSCs, their role in the pathophysiology of joint diseases and provide the basis for new avenues for treatment.

Stem cells combined with bone graft substitutes in skeletal tissue engineering.

INTRODUCTION: Bone grafting is used to repair large bone defects and autograft is recognised as producing the best clinical outcome, which is partly due to its cellular component. When autograft is unavailable, allograft and bone graft substitutes can be used; however, they rely on the host bed to provide cellular osteogenic activity. AREAS COVERED: Bone graft substitutes have the potential to benefit from the addition of stem cells aimed at enhancing the rate and quality of defect repair. Mesenchymal stem cells (MSCs) can be isolated from bone marrow or periosteum and culture expanded. Other sources of primary cells include muscle, adipose tissue, human umbilical cord and the pluripotent embryonic stem cells (ESCs). EXPERT OPINION: MSCs isolated from bone marrow have been the best characterised approach for osteogenic differentiation. Their use with synthetic scaffolds such as hydroxyapatite and tricalcium phosphate has produced promising clinical results. MSCs derived from adipose tissue, muscle or human umbilical cord cells combined with various scaffolds are an attractive option. Further in vivo and clinical investigation of their potential is required. Pluripotent ESCs have a theoretical advantage over MSCs; however, purification, cell-specific differentiation, effective delivery vehicles-scaffolds and teratogenesis control are still under in vitro and in vivo evaluation.

Role of robotic surgery in oral and maxillofacial, and head and neck surgery.

We review the current status of robotic surgery in the head and neck region and its role in oral and maxillofacial surgery.

Prostaglandin E(2) receptors as potential bone anabolic targets - selective EP4 receptor agonists.

Prostaglandin E(2) is known to be a potent metabolite in bone biology. Its effects are mediated via four receptor subtypes with different properties, effects and mechanisms of action. The EP2 and EP4 receptors have been extensively investigated as bone anabolic therapy targets in the literature. The aim of this review was to analyse the available evidence supporting the use of selective agonists for those receptors for anabolic bone application purposes. Although several studies report on the presence of the EP2 receptor in several cell types, efforts to directly confirm the presence of this receptor in human bone cells have not been successful. The EP4 receptor however has been identified in human bone cells and its significant role in bone biology has been demonstrated with the use of selective agonists, antagonists and transgenic small animals. The use of selective EP4 agonists reversed established osteoporotic changes, enhanced the boneimplant interface strength and was shown to have a synergistic effect when used with other bone cell targeting pharmacological agents such as BMP-2 and bisphosphonates. Further elucidation of the side-effect profile of prostanoid and non-prostanoid agonists is required for these agents to proceed towards clinical applications.

In vitro and in vivo effects of antibiotics on bone cell metabolism and fracture healing.

INTRODUCTION: Recent evidence suggests that antibiotics exert direct effects on bone at a cellular level, disrupting mitochondrial function and cell activity. This comprehensive literature review aims to evaluate evidence for the effects of antibiotics and antimicrobials on bone and discuss the clinical implications. AREAS COVERED: A literature search was conducted on electronic databases covering a period from 1969 to 2010. Studies were included if they reported in vivo and in vitro experimental findings regarding the use of antibiotics and synthetic antibacterials in both animals and humans, focusing on bone cell function and especially fracture repair. EXPERT OPINION: Current research suggests that these negative results could be due to direct effects of antibiotics on mitochondrial physiology within mammalian cells. Treatment doses of antibiotics, especially those released from topical delivery systems such as bone cements, result in antibiotic concentrations thousands of times higher than those required to inhibit bacterial growth. Our findings suggest a need to develop current antibiotic delivery systems to elute sufficient doses to inhibit bacterial growth without negative effects on bone physiology and fracture repair processes.

Risk of osteoporosis and fracture incidence in patients on antipsychotic medication.

INTRODUCTION: In patients suffering from schizophrenia and bipolar disorder, antipsychotics are the mainstay of treatment worldwide. By blocking D(2) brain mesolimbic receptors, antipsychotics are believed to reduce and control psychotic experiences, but recent evidence has suggested that they may also have adverse effects on bone mineral architecture and fracture incidence. AREAS COVERED: This study reviews current literature surrounding the use of antipsychotics and their effects on bone homeostasis. The primary medical search engines used for the study are Ovid MEDLINE (1950 - April 2010), EMBASE (1988 - April 2010) and PsychINFO (1987 - April 2010) databases. EXPERT OPINION: Typical antipsychotics, in addition to the atypical antipsychotics risperidone and amisulpride, have been shown to increase serum prolactin levels in in vivo human studies. Results from animal and human in vitro and in vivo studies have demonstrated that high concentrations of prolactin have been shown to adversely affect bone cell metabolism and accelerate the rate bone mineral density loss, thereby increasing fracture risk. Increasing awareness of the side effect profile of antipsychotic medications on bone metabolism may prompt clinicians to screen patients at high risk of antipsychotic-induced osteoporosis and provide treatment, which may reduce the incidence of potentially avoidable fractures.

Perspectives in regenerative medicine and tissue engineering of bone.

This final review in a series of three focuses on how the advances made in the realm of molecular and cellular bone biology have been translated into experimental animal and human surgery.

Simvastatin induces osteogenic differentiation of murine embryonic stem cells.

Statins are potent inhibitors of cholesterol synthesis. Several statins are available with different molecular and pharmacokinetic properties. Simvastatin is more lipophilic than pravastatin and has a higher affinity to phospholipid membranes than atorvastatin, allowing its passive diffusion through the cell membrane. In vitro studies on bone marrow stromal cells, osteoblast-like cells, and embryonic stem cells have shown statins to have cholesterol-independent anabolic effects on bone metabolism; alas, statins were supplemented in osteogenic medium, which does not facilitate elucidation of their potential osteoinductive properties. Embryonic stem cells (ESCs), derived from the inner cell mass of the blastocyst, are unique in that they enjoy perpetual self-proliferation, are pluripotent, and are able to differentiate toward all the cellular lineages composing the body, including the osteogenic lineage. Consequently, ESCs represent a potentially potent cell source for future clinical cellular therapies of various bone diseases, even though there are several hurdles that still need to be overcome. Herein we demonstrate, for the first time to our knowledge, that simvastatin induces murine ESC (mESC) differentiation toward the osteogenic lineage in the absence of osteoinductive supplements. Specifically, we found that a simvastatin concentration in the micromolar range and higher was toxic to the cells and that an effective concentration for osteoinduction is 0.1 nM, as shown by increased alizarin red staining as well as increased osteocalcin and osetrix gene expression. These results suggest that in the future, lipophilic simvastatin may provide a novel pharmacologic agent for bone tissue engineering applications.

Osteoinduction: translating preclinical promise into clinical reality.

This review, the second in a series of three editorials, focuses on the problems of translating basic scientific research on induction of bone into reliable clinical applications.

Biological therapy of bone defects: the immunology of bone allo-transplantation.

IMPORTANCE OF THE FIELD: Bone is one of the most transplanted tissues worldwide. Autograft is the ideal bone graft but is not widely used because of donor site morbidity and restricted availability. Allograft is easily accessible but can transmit infections and elicit an immune response. AREAS COVERED IN THIS REVIEW: This review identifies all in vitro and in vivo evidence of immune responses following bone transplantation and highlights methods of improving host tolerance to bone allotransplantation. WHAT THE READER WILL GAIN: In humans, the presence of anti-HLA specific antibodies against freeze-dried and fresh-frozen bone allografts has been demonstrated. Fresh-frozen bone allograft can still generate immune reactions whilst freeze-dried bone allografts present with less immunogenicity but have less structural integrity. This immune response can have an adverse effect on the graft's incorporation and increase the incidence of rejection. Decreasing the immune reaction against the allograft by lowering the immunogenic load of the graft or lowering the host immune response, would result in improved bone incorporation. TAKE HOME MESSAGE: It is essential that the complex biological processes related to bone immunogenicity are understood, since this may allow the development of safer and more successful ways of controlling the outcome of bone allografting.