Endogenous FGF-2 levels impact FGF-2/BMP-2 growth factor delivery dosing in aged murine calvarial bone defects.

Bone repair in elderly mice has been shown to be improved or negatively impacted by supplementing the highly osteogenic bone morphogenetic protein-2 (BMP-2) with fibroblast growth factor-2 (FGF-2). To better predict the outcome of FGF-2 supplementation, we investigated whether endogenous levels of FGF-2 play a role in optimal dosing of FGF-2 for augmenting BMP-2 activity in elderly mice. In vivo calvarial bone defect studies in Fgf2 knockout mice with wildtype controls were conducted with the growth factors delivered in a highly localized manner from a biomimetic calcium phosphate/polyelectrolyte multilayer coating applied to a bone graft substitute. Endogenous FGF-2 levels were measured in old mice versus young and found to decrease with age. Optimal dosing for improving bone defect repair correlated with levels of endogenous FGF-2, with a larger dose of FGF-2 required to have a positive effect on bone healing in the Fgf2 knockout mice. The same dose in wildtype old mice, with higher levels of FGF-2, promoted chondrogenesis and increased osteoclast activity. The results suggest a personalized medicine approach, based on a knowledge of endogenous levels of FGF-2, should guide FGF-2 supplementation in order to avoid provoking excessive bone resorption and cartilage formation, both of which inhibited calvarial bone repair.

Cell Type Influences Local Delivery of Biomolecules from a Bioinspired Apatite Drug Delivery System.

Recently, the benefit of step-wise sequential delivery of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 from a bioinspired apatite drug delivery system on mouse calvarial bone repair was demonstrated. The thicknesses of the nanostructured poly-l-Lysine/poly-l-Glutamic acid polyelectrolyte multilayer (PEM) and the bone-like apatite barrier layer that make up the delivery system, were varied. The effects of the structural variations of the coating on the kinetics of cell access to a cytotoxic factor delivered by the layered structure were evaluated. FGF-2 was adsorbed into the outer PEM, and cytotoxic antimycin-A (AntiA) was adsorbed to the substrate below the barrier layer to detect the timing of the cell access. While MC3T3-E1 osteoprogenitor cells accessed AntiA after three days, the RAW 264.7 macrophage access occurred within 4 h, unless the PEM layer was removed, in which case the results were reversed. Pits were created in the coating by the RAW 264.7 macrophages and initiated delivery, while the osteoprogenitor cell access to drugs occurred through a solution-mediated coating dissolution, at junctions between the islands of crystals. Macrophage-mediated degradation is therefore a mechanism that controls drug release from coatings containing bioinspired apatite.

* Calvarial Bone Regeneration Is Enhanced by Sequential Delivery of FGF-2 and BMP-2 from Layer-by-Layer Coatings with a Biomimetic Calcium Phosphate Barrier Layer.

A drug delivery coating for synthetic bone grafts has been developed to provide sequential delivery of multiple osteoinductive factors to better mimic aspects of the natural regenerative process. The coating is composed of a biomimetic calcium phosphate (bCaP) layer that is applied to a synthetic bone graft and then covered with a poly-l-Lysine/poly-l-Glutamic acid polyelectrolyte multilayer (PEM) film. Bone morphogenetic protein-2 (BMP-2) was applied before the coating process directly on the synthetic bone graft and then, bCaP-PEM was deposited followed by adsorption of fibroblast growth factor-2 (FGF-2) into the PEM layer. Cells access the FGF-2 immediately, while the bCaP-PEM temporally delays the cell access to BMP-2. In vitro studies with cells derived from mouse calvarial bones demonstrated that Sca-1 and CD-166 positive osteoblast progenitor cells proliferated in response to media dosing with FGF-2. Coated scaffolds with BMP-2 and FGF-2 were implanted in mouse calvarial bone defects and harvested at 1 and 3 weeks. After 1 week in vivo, proliferation of cells, including Sca-1+ progenitors, was observed with low dose FGF-2 and BMP-2 compared to BMP-2 alone, indicating that in vivo delivery of FGF-2 activated a similar population of cells as shown by in vitro testing. At 3 weeks, FGF-2 and BMP-2 delivery increased bone formation more than BMP-2 alone, particularly in the center of the defect, confirming that the proliferation of the Sca-1 positive osteoprogenitors by FGF-2 was associated with increased bone healing. Areas of bone mineralization were positive for double fluorochrome labeling of calcium and alkaline phosphatase staining of osteoblasts, along with increased TRAP+ osteoclasts, demonstrating active bone formation distinct from the bone-like collagen/hydroxyapatite scaffold. In conclusion, the addition of a bCaP layer to PEM delayed access to BMP-2 and allowed the FGF-2 stimulated progenitors to populate the scaffold before differentiating in response to BMP-2, leading to improved bone defect healing.

Human biofield therapy does not affect tumor size but modulates immune responses in a mouse model for breast cancer.

OBJECTIVE: To assess the effect of human biofield therapy, an integrative medicine modality, on the development of tumors and metastasis, and immune function in a mouse breast cancer model. METHODS: Mice were injected with 66cl4 mammary carcinoma cells. In study one, mice received biofield therapy after cell injection. In study two, mice were treated by the biofield practitioner only prior to cell injection. Both studies had two control groups of mock biofield treatments and phosphate-buffered saline injection. Mice were weighed and tumor volume was determined. Blood samples were collected and 32 serum cytokine/chemokine markers were measured. Spleens/popliteal lymph nodes were isolated and dissociated for fluorescent-activated cell sorting (FACS) analysis of immune cells or metastasis assays in cell culture. RESULTS: No significant differences were found in weight, tumor size or metastasis. Significant effects were found in the immune responses in study one but no additional effects were found in study two. In study one, human biofield treatment significantly reduced percentage of CD4(+)CD44loCD25(+) and percentage of CD8(+) cells, elevated by cancer in the lymph nodes, to control levels determined by FACS analysis. In the spleen, only CD11b(+) macrophages were increased with cancer, and human biofield therapy significantly reduced them. Of 11 cytokines elevated by cancer, only interferon-γ, interleukin-1, monokine induced by interfer-γ, interleukin-2 and macrophage inflammatory protein-2 were significantly reduced to control levels with human biofield therapy. CONCLUSION: Human biofield therapy had no significant effect on tumor size or metastasis but produced significant effects on immune responses apparent in the down-regulation of specific lymphocytes and serum cytokines in a mouse breast cancer model.

Age-Related Changes in FGF-2, Fibroblast Growth Factor Receptors and ß-Catenin Expression in Human Mesenchyme-Derived Progenitor Cells.

FGF-2 stimulates preosteoblast replication, and knockout of the FGF-2 gene in mice resulted in osteopenia with age, associated with decreased Wnt-ß-Catenin signaling. In addition, targeted expression of FGF-2 in osteoblast progenitors increased bone mass in mice via Wnt-ß-Catenin signaling. We posited that diminution of the intrinsic proliferative capacity of human mesenchyme-derived progenitor cells (HMDPCs) with age is due in part to reduction in FGF-2. To test this hypothesis HMDPCs from young (27-38), middle aged (47-56), and old (65-76) female human subjects were isolated from bone discarded after orthopedic procedures. HMDPCs cultures were mostly homogeneous with greater than 90% mesenchymal progenitor cells, determined by fluorescence-activated cell sorting. There was a progressive decrease in FGF-2 and FGFR1 mRNA and protein in HMDPCs with age. Since FGF-2 activates ß-catenin, which can enhance bone formation, we also assessed its age-related expression in HMDPCs. An age-related decrease in total-ß-Catenin mRNA and protein expression was observed. However there were increased levels of p-ß-Catenin and decreased levels of activated-ß-Catenin in old HMDSCs. FGF-2 treatment increased FGFR1 and ß-Catenin protein, reduced the level of p-ß-Catenin and increased activated-ß-Catenin in aged HMDPCs. In conclusion, reduction in FGF-2 expression could contribute to age-related impaired function of HMDPCs via modulation of Wnt-ß-catenin signaling.

Challenges for Preclinical Investigations of Human Biofield Modalities.

Preclinical models for studying the effects of the human biofield have great potential to advance our understanding of human biofield modalities, which include external qigong, Johrei, Reiki, therapeutic touch, healing touch, polarity therapy, pranic healing, and other practices. A short history of Western biofield studies using preclinical models is presented and demonstrates numerous and consistent examples of human biofields significantly affecting biological systems both in vitro and in vivo. Methodological issues arising from these studies and practical solutions in experimental design are presented. Important questions still left unanswered with preclinical models include variable reproducibility, dosing, intentionality of the practitioner, best preclinical systems, and mechanisms. Input from the biofield practitioners in the experimental design is critical to improving experimental outcomes; however, the development of standard criteria for uniformity of practice and for inclusion of multiple practitioners is needed. Research in human biofield studies involving preclinical models promises a better understanding of the mechanisms underlying the efficacy of biofield therapies and will be important in guiding clinical protocols and integrating treatments with conventional medical therapies.

Therapeutic Touch Has Significant Effects on Mouse Breast Cancer Metastasis and Immune Responses but Not Primary Tumor Size.

Evidence-based integrative medicine therapies have been introduced to promote wellness and offset side-effects from cancer treatment. Energy medicine is an integrative medicine technique using the human biofield to promote well-being. The biofield therapy chosen for study was Therapeutic Touch (TT). Breast cancer tumors were initiated in mice by injection of metastatic 66cl4 mammary carcinoma cells. The control group received only vehicle. TT or mock treatments were performed twice a week for 10 minutes. Two experienced TT practitioners alternated treatments. At 26 days, metastasis to popliteal lymph nodes was determined by clonogenic assay. Changes in immune function were measured by analysis of serum cytokines and by fluorescent activated cells sorting (FACS) of immune cells from the spleen and lymph nodes. No significant differences were found in body weight gain or tumor size. Metastasis was significantly reduced in the TT-treated mice compared to mock-treated mice. Cancer significantly elevated eleven cytokines. TT significantly reduced IL-1-a, MIG, IL-1b, and MIP-2 to control/vehicle levels. FACS demonstrated that TT significantly reduced specific splenic lymphocyte subsets and macrophages were significantly elevated with cancer. Human biofield therapy had no significant effect on primary tumor but produced significant effects on metastasis and immune responses in a mouse breast cancer model.

Effects of low dose FGF-2 and BMP-2 on healing of calvarial defects in old mice.

There is an age-associated reduction in the bone healing activity of bone morphogenetic protein-2 (BMP-2) that is currently addressed by administering higher doses of BMP-2 in elderly patients. The unwanted medical complications from high dose BMP-2 motivated this investigation to determine whether the addition of a low dose of fibroblast growth factor 2 (FGF-2) could enhance the ability of a lower dose of BMP-2 to heal calvarial bone defects in old mice (18-20 months old). FGF-2 (5 ng) and BMP-2 (2 µg) were administered by a controlled release two-phase biomaterial scaffold placed into the bone defect. FGF-2 released more rapidly and completely in vitro than BMP-2 (40% vs 2%). In vivo, both BMP-2 and FGF-2+BMP-2 groups formed more new bone in calvarial defects than scaffold alone (p < 0.001) or FGF-2 only groups (p < 0.01). The overall total volume of new bone was not statistically increased by the addition of FGF-2 to BMP-2 as measured by microCT, but the pattern of bone deposition was different. In old mice, but not young, there was enhanced bony fill in the central bone defect area when the BMP-2 was supplemented with FGF-2. Histological analysis of the center of the defect revealed an increased bone volume (%BV/TV (p = 0.004)) from the addition of FGF-2. These studies suggest that combining a low dose of FGF-2 with a low dose of BMP-2 has the potential to increase bone healing in old mice relative to BMP-2 alone.

Chronic rhinosinusitis osteoblasts differ in cellular properties from normal bone.

BACKGROUND: Osteitis, characterized by bony thickening and remodeling, is often considered a hallmark of recalcitrant rhinosinusitis. However, there is limited literature examining the bone in chronic rhinosinusitis (CRS) pathology. In this study we cultured osteoblasts from bone harvested during sinus surgery as well as from nondiseased controls to compare their cellular properties. METHODS: Sinus bone was collected during sinus and skull-base surgery and placed in proliferation media. Outgrowth of cells occurred at 2 weeks and the cells were confirmed to be osteoblasts by alkaline phosphatase staining. Cellular adhesion was determined by replating and counting adhered cells at 4 hours. Proliferation of cells plated for 24 hours was assayed by measuring [(3) H]-thymidine incorporation. Calcium content was measured by changing cells to differentiation media and measuring the calcium content on days 7, 14, and 21. RESULTS: Alkaline phosphatase assay showed more than 90% of osteoblasts staining in all samples. Osteoblasts from patients with CRS had significant decreases in adhesion (p < 0.01) compared to osteoblasts from skull-base patients. There was a significant (p < 0.05) increase in calcium content in rhinosinusitis samples compared with the nondiseased sinus bone samples. CONCLUSION: To date, this is the first known study that shows a direct comparison of osteoblast properties between patients with and without CRS. Our results indicate that there are fundamental phenotypic differences in adhesion and mineralization between osteoblasts in patients with CRS compared to controls.

Differences in otosclerotic and normal human stapedial osteoblast properties are normalized by alendronate in vitro.

OBJECTIVE: Identify and compare phenotypic properties of osteoblasts from patients with otosclerosis (OSO), normal bones (HOB), and normal stapes (NSO) to determine a possible cause for OSO hypermineralization and assess any effects of the bisphosphonate, alendronate. STUDY DESIGN: OSO (n = 11), NSO (n = 4), and HOB (n = 13) cultures were assayed for proliferation, adhesion, mineralization, and gene expression with and without 10(-10)M-10(-8)M alendronate. SETTING: Academic hospital. METHODS: Cultures were matched for age, sex, and passage number. Cell attachment and proliferation + alendronate were determined by Coulter counting cells and assaying tritiated thymidine uptake, respectively. At 7, 14, and 21 days of culture + alendronate, calcium content and gene expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were determined. RESULTS: OSO had significantly more cells adhere but less proliferation than NSO or HOB. Calcification was significantly increased in OSO compared to HOB and NSO. NSO and HOB had similar cell adhesion and proliferation rates. A dose-dependent effect of alendronate on OSO adhesion, proliferation, and mineralization was found, resulting in levels equal to NSO and HOB. All cultures expressed osteoblast-specific genes such as RUNX2, alkaline phosphatase, type I collagen, and osteocalcin. However, osteopontin was dramatically reduced, 9.4-fold at 14 days, in OSO compared to NSO. Receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG), important in bone resorption, was elevated in OSO with decreased levels of OPG levels. Alendronate had little effect on gene expression in HOB but in OSO increased osteopontin levels and decreased RANKL/OPG. CONCLUSIONS: OSO cultures displayed properties of hypermineralization due to decreased osteopontin (OPN) and also had increased RANKL/OPG, which were normalized by alendronate.

Fibroblast growth factor-2 and bone morphogenetic protein-2 have a synergistic stimulatory effect on bone formation in cell cultures from elderly mouse and human bone.

Combined regimens of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) were investigated to stimulate osteogenic differentiation. In young mouse calvaria-derived cells, FGF-2 (0.16ng/mL) in combination with BMP-2 (50ng/mL) did not enhance mineralization, but in old mouse cells it resulted in more mineralization than BMP-2 alone. In young long bone mouse cultures, FGF-2 enhanced mineralization relative to BMP-2 alone, but in old cultures, lower dose of FGF-2 (0.016ng/mL) was necessary. In neonatal mouse calvarial cells, sequential delivery of low-dose FGF-2 and low-dose BMP-2 (5ng/mL) was more stimulatory than co-delivery. In young human cultures, 0.016ng/mL of FGF-2 did not enhance mineralization, in combination with 5ng/mL of BMP-2, but in older cultures, codelivery of FGF-2 and BMP-2 was superior to BMP-2 alone. In conclusion, BMP-2 treatment alone was sufficient for maximal mineralization in young osteoblast cultures. However, coadministration of FGF-2 and BMP-2 increases mineralization more than BMP-2 alone in cultures from old and young mouse long bones and old humans but not in young mouse calvarial cultures.

Fibroblast growth factor-2, bone homeostasis and fracture repair.

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.

One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating.

Controlled differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into cells that resemble adult mesenchymal stem cells (MSCs) is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. The present study reports a new method to rapidly derive MSC-like cells from hESCs and hiPSCs, in one step, based on culturing the cells on thin, fibrillar, type I collagen coatings that mimic the structure of physiological collagen. Human H9 ESCs and HDFa-YK26 iPSCs were singly dissociated in the presence of ROCK inhibitor Y-27632, plated onto fibrillar collagen coated plates and cultured in alpha minimum essential medium (alpha-MEM) supplemented with 10% fetal bovine serum, 50 uM magnesium L-ascorbic acid phosphate and 100 nM dexamethasone. While fewer cells attached on the collagen surface initially than standard tissue culture plastic, after culturing for 10 days, resilient colonies of homogenous spindle-shaped cells were obtained. Flow cytometric analysis showed that a high percentage of the derived cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146 and CD166 and were negative as expected for hematopoietic markers CD34 and CD45. The MSC-like cells derived from pluripotent cells were successfully differentiated in vitro into three different lineages: osteogenic, chondrogenic, and adipogenic. Both H9 hES and YK26 iPS cells displayed similar morphological changes during the derivation process and yielded MSC-like cells with similar properties. In conclusion, this study demonstrates that bioimimetic, fibrillar, type I collagen coatings applied to cell culture plates can be used to guide a rapid, efficient derivation of MSC-like cells from both human ES and iPS cells.

New insights on therapeutic touch: a discussion of experimental methodology and design that resulted in significant effects on normal human cells and osteosarcoma.

Our purpose is to discuss the study design and innovative approaches that led to finding significant effects of one energy medicine therapy, Therapeutic Touch (TT), on cells. In the original published studies, TT was shown to significantly increase human osteoblast DNA synthesis, differentiation, and mineralization; increase in a dose-dependent manner the growth of other human cell types; and decrease the differentiation and mineralization of a human osteosarcoma-derived cell line. A unique feature of the study's methodology and design that contributed to the success of the findings was that a basic level of skill and maturity of the TT practitioner was quantified for producing observable and replicable outcomes in a test administered to all TT practitioners. Only those practitioners that passed the test were selected for the study. (2) The practitioners were required to keep a journal, which appeared to promote their ability to stay centered and replicate their treatments over months of cell experimentation. (3) The origin of the cells that the practitioners were treating was explained to them, although they were blinded to cell type during the experiments. (4) Only early passage cells were used to maintain a stable cell phenotype. (5) Standard protocols for performing TT in the room were followed to ensure reproducible conditions. (6) Placebo controls and untreated controls were used for each experiment. (7) The principal investigator and technicians performing the assays were blinded as to the experimental groups, and all assays and procedures were well established in the laboratory prior to the start of the TT experiments. The absence of studies on the human biofield from mainstream scientific literature is also discussed by describing the difficulties encountered in publishing. These roadblocks contribute to our lack of understanding of the human biofield and energy medicine modalities in science. In conclusion, this report seeks to encourage well-designed, evidence-based studies on the human biofield and the therapeutic potential of the human biofield.

Porous tantalum stimulates the proliferation and osteogenesis of osteoblasts from elderly female patients.

Porous tantalum (Ta) implants have been successful in various orthopedic procedures for patients with compromised bone-forming abilities. Previous studies demonstrated that human osteoblast (HOB) cultures from older female patients produced less bone on implant materials in vitro compared to HOBs from age-matched male and younger female patients. In this study, the responses of HOBs from younger (< 45) and older (> 60 years old) female patients were compared on Ta, titanium fiber mesh (TFM) and tissue culture plastic. Adhesion, proliferation, and mineralization were greater in cells from younger patients than from older patients. Cell adhesion was slightly higher on Ta than TFM or plastic. However, Ta highly stimulated cell proliferation with a 4- and 6-fold increase compared to TFM for cells from younger and older patients, respectively, and 12- and 16-fold increase in proliferation compared to cells on plastic (p ≤ 0.001). At 3 weeks, mineralization was significantly higher on Ta compared to TFM for HOBs from older patients (p ≤ 0.05). Expression levels of bone matrix markers demonstrated differences dependent on age and substrate. Scanning electron micrographs revealed HOBs covering the surfaces and entering the pores of both Ta and TFM. In conclusion, tantalum greatly stimulates cell proliferation, and improves the ability of HOBs from older patients to form bone.

Fibroblast growth factor-2 stimulates the proliferation of mesenchyme-derived progenitor cells from aging mouse and human bone.

The potential of fibroblast growth factor-2 (FGF-2) to stimulate osteoprogenitors in aging bone was investigated. Previous work showed a decrease in bone formation in cell cultures derived from bone of elderly female patients, but not in cells from age-matched male or younger female patients, with transforming growth factor ß increasing bone formation but not increasing osteoprogenitors. In the present study, FGF-2 was shown to significantly stimulate, in a dose-dependent manner, proliferation of mesenchyme-derived progenitor cells from bones of young and old mouse and humans. In proliferation assays, human cells were more responsive to lower concentrations (0.0016 ng/mL) of FGF-2 than mouse cells, but proliferation was less in cells from older bone. Immunofluorescence microscopy revealed that FGF-2 increased and prevented the decline in cells expressing activated leukocyte cell adhesion molecule, a novel marker for early lineage osteoblasts, but not α-smooth muscle actin. FGF-2 may have therapeutic potential for stimulating osteoblast progenitors in aging.

Fabrication and characterization of hydroxyapatite-coated polystyrene disks for use in osteoprogenitor cell culture.

A simple method is reported for fabricating polystyrene disk inserts coated with biomimetic carbonated hydroxyapatite (cHA) to be used for culturing osteoprogenitor cells or other stem cells. Roughened disks cut from tissue-culture polystyrene (TCPS) were coated in simulated body fluid with 5 x normal physiologic ionic concentrations (SBFx5) by a 2-step, 2-day method. The coatings were rigorously characterized by various methods and assessed in cell culture. An adherent, nearly 10 mm thick, relatively uniform layer of single-phase cHA was formed in two days. MC3T3-E1 and mouse calvaria-derived osteoprogenitor cells (pCOBs) were cultured on the cHA for various time points. Despite less initial attachment of both cell types to the cHA, proliferation rates on cHA were similar to that on TCPS. Two-fold greater cell attachment (P < 0.05) of the MC3T3-E1 cells was observed relative to the pCOBs, on both the TCPS and the cHA. Importantly, the coatings were relatively smooth, without the extensive agglomerates observed in other studies and remained adherent and morphologically unchanged after 21 days of culture. This technique can be used to rapidly produce high-quality cHA-coated TCPS disks for cell-culture studies.