Manolis Heliotis

TGF-β3: A potential biological therapy for enhancing chondrogenesis

Background: TGF-β has been proposed to stimulate chondrogenesis through intracellular pathways involving small mothers against decapentaplegic proteins (Smads). Objective: To examine the use of exogenous TGF-β3 to promote new hyaline cartilage formation. Methods: An overview of in vitro and in vivo evidence on the effects of TGF-β3 on cartilage regeneration. Results/conclusion: There is robust in vitro evidence suggesting a positive dose- and time-dependant effect of TGF-β3 on anabolic chondrogenic gene markers such as α1-collagen type II and cartilage oligomeric matrix protein in human mesenchymal stem cells. TGF-β3 cultured with silk elastin-like polymer scaffold carrier exhibits significantly increased glycosaminoglycan and collagen content. In vivo data showed that TGF-β3 cultured with ovine mesenchymal stem cells in a chitosan scaffold stimulated the growth of hyaline cartilage that was fully integrated into host cartilage tissue of sheep. We highlight the potential for the clinical enhancement of cartilage formation through the use of TGF-β3 with a suitable dose and scaffold carrier.

Investigating the role of PDGF as a potential drug therapy in bone formation and fracture healing

Background: Platelet-derived growth factor (PDGF) has been shown in vivo to increase bone formation and supplement fracture healing, and may have a role as a therapeutic agent in the treatment of bone loss and fracture healing in humans. Objective: A comprehensive review of the recent literature on the effect of PDGF on bone mineral density and fracture healing. Methods: In vitro and in vivo evidence was systematically collected using medical search engines MEDLINE/OVID (1950 to March 2008) and EMBASE (1980 to March 2008) databases. Results/conclusion: Evidence to date suggests that PDGF-BB, and to a lesser extent PDGF-AA, may have potential therapeutic use in the treatment of osteoporosis and bone healing in humans. Additionally, by targeting α-receptors on osteoblasts, a potential anabolic effect on bone metabolism in humans can be anticipated; however, more research needs to be done to assess the role of β-receptors in human bone.

Soluble and insoluble signals and the induction of bone formation: molecular therapeutics recapitulating development

The osteogenic molecular signals of the transforming growth factor‐β (TGF‐β) superfamily, the bone morphogenetic/osteogenic proteins (BMPs/OPs) and uniquely in primates the TGF‐β isoforms per se, pleiotropic members of the TGF‐β supergene family, induce de novo endochondral bone formation as a recapitulation of embryonic development. Naturally derived BMPs/OPs and gamma‐irradiated human recombinant osteogenic protein‐1 (hOP‐1) delivered by allogeneic and xenogeneic insoluble collagenous matrices initiate de novo bone induction in heterotopic and orthotopic sites of the primate Papio ursinus, culminating in complete calvarial regeneration by day 90 and maintaining the regenerated structures by day 365. The induction of bone by hOP‐1 in P. ursinus develops as a mosaic structure with distinct spatial and temporal patterns of gene expression of members of the TGF‐β superfamily that singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis. The temporal and spatial expressions of TGF‐β1 mRNA indicate a specific temporal transcriptional window during which expression of TGF‐β1 is mandatory for successful and optimal osteogenesis. Highly purified naturally derived bovine BMPs/OPs and hOP‐1 delivered by human collagenous bone matrices and porous hydroxyapatite, respectively, induce bone formation in mandibular defects of human patients. By using healthy body sites as bioreactors it is possible to recapitulate embryonic developments by inducing selected biomaterials combined with recombinant proteins to transform into custom‐made prefabricated bone grafts for human reconstruction. The osteogenic proteins of the TGF‐β superfamily, BMPs/OPs and TGF‐βs, the last endowed with the striking prerogative of inducing endochondral bone formation in primates only, are helping to engineer skeletal reconstruction in molecular terms.

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.

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.

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 grafts 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.

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.

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 D2 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.

Targeting bone morphogenetic protein antagonists: in vitro and in vivo evidence of their role in bone metabolism

Background: Bone morphogenetic proteins (BMPs) and their antagonists are involved in fracture healing. Antagonists regulate BMPs by blocking signal transduction or interfering with transcription factors at the nucleus. Objective: To examine targeting of BMP antagonists to manipulate osteogenesis. Methods: An overview of in vitro and in vivo evidence on effects of BMP antagonists on bone metabolism. Results/conclusion: There is in vitro evidence suggesting that overexpression of noggin and gremlin inhibits osteogenic differentiation, markedly decreases alkaline phosphatase (ALP) levels and impedes R-Smad (1/5/8) phosphorylation in murine cell lines. Knockdown of chordin results in a threefold increase in ALP activity in human mesenchymal stem cells. In vivo data shows that inhibition of noggin leads to increased bone regeneration in mice. Noggin and sclerostin can combine in a mutually inhibitory complex, neutralising their individual inhibitory effects. This allows BMP signalling to proceed to osteoinduction. We highlight the potential for clinical enhancement of bone formation through inhibition of BMP antagonists.

Prostaglandin EP2 and EP4 receptor agonists in bone formation and bone healing: In vivo and in vitro evidence

Background: Using agonists that selectively stimulate PGE2 receptors, the adverse effects that have limited the clinical utility of PGE2 can be avoided and there may be potential for their use as therapeutic agents in the treatment of bone loss in humans. Objective: A comprehensive review of the recent literature on the effect of prostaglandins and their agonists on bone mineral density and fracture healing. Methods: In vitro and in vivo evidence was collected using medical search engines MEDLINE® (1950 to March 2008) and EMBASE (1980 to March 2008) databases. Results/conclusion: EP4 receptors have been identified in human osteoblast cell lines and have also been shown to activate osteoblast directly and osteoclast indirectly via osteoblastic activation. Although there are strong in vitro and in vivo collective data indicating that EP2 receptors may have a role in mediating the anabolic effects of PGE2 on bone, to date no functional EP2 receptors have been identified on human osteoblasts or osteoclasts. This suggests that PGE2 effect on bone formation and resorption in humans may be governed by activation of the EP4 receptor on osteoblasts. Selective EP4 receptor agonists may therefore provide therapeutic potential for systemic use in the treatment of osteoporosis and fracture healing. Further studies need to be carried out in order fully elicit the role of EP2 receptor agonists in fracture healing and bone formation in humans.