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.

miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop.

Differentiation of human mesenchymal stem cells into osteoblasts is controlled by extracellular cues. Canonical Wnt signaling is particularly important for maintenance of bone mass in humans. Post-transcriptional regulation of gene expression, mediated by microRNAs, plays an essential role in the control of osteoblast differentiation. Here, we find that miR-29a is necessary for human osteoblast differentiation, and miR-29a is increased during differentiation in the mesenchymal precursor cell line hFOB1.19 and in primary cultures of human osteoblasts. Furthermore, the promoter of the expressed sequence tag containing the human miR-29a gene is induced by canonical Wnt signaling. This effect is mediated, at least in part, by two T-cell factor/LEF-binding sites within the proximal promoter. Furthermore, we show that the negative regulators of Wnt signaling, Dikkopf-1 (Dkk1), Kremen2, and secreted frizzled related protein 2 (sFRP2), are direct targets of miR-29a. Endogenous protein levels for these Wnt antagonists are increased in cells transfected with synthetic miR-29a inhibitor. In contrast, transfection with miR-29a mimic decreases expression of these antagonists and potentiates Wnt signaling. Overall, we demonstrate that miR-29 and Wnt signaling are involved in a regulatory circuit that can modulate osteoblast differentiation. Specifically, canonical Wnt signaling induces miR-29a transcription. The subsequent down-regulation of key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, by miR-29a potentiates Wnt signaling, contributing to a gene expression program important for osteoblast differentiation. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during osteoblast differentiation, allowing for fine-tuning of intricate cellular processes.

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.

Odontoblast-targeted Bcl-2 overexpression impairs dentin formation.

Apoptosis has been described extensively in tooth development, which is under tight control of multiple apoptosis regulators, including anti-apoptotic protein Bcl-2. However, it is totally unclear how Bcl-2 is related to odontogenesis, especially dentinogenesis. Using a transgenic mouse Col2.3Bcl-2 in which human Bcl-2 was overexpressed in odontoblasts, the effect of Bcl-2 on dentinogenesis was investigated. Overexpression of Bcl-2 was detected by immunohistochemistry and Western blot. Odontoblast apoptosis was evaluated by TUNEL and Western blot detection of cleaved caspase-3. Odontoblast differentiation was assessed by real-time PCR detection of dentin matrix expression. Dentin mineralization was evaluated by micro-CT in vivo, and alizarin red S staining and calcium content analysis in vitro. Bcl-2 was found to be overexpressed in odontoblasts and prevent their apoptosis. Odontoblast differentiation and mineralization was inhibited by Bcl-2, as evidenced by lower expressions of DMP-1, OC, and DSPP, and decreased odontoblast mineralization in vitro, as well as decreased dentin thickness and mineral density in vivo when compared to the wild-type animals. Inhibition of odontoblast differentiation by Bcl-2 occurs, at least partially, via a suppression of MEK-ERK1/2 signaling pathway. In conclusion, Bcl-2 overexpression prevents odontoblast apoptosis and impairs dentin formation, partially via an inhibition of odontoblast differentiation. This study revealed some novel functions of Bcl-2 in dentinogenesis in addition to its anti-apoptotic effect, which shed some light on the genetic complexity of tooth development.

Osteopenia in transgenic mice with osteoblast-targeted expression of the inducible cAMP early repressor.

ICER is a member of the CREM family of basic leucine zipper transcription factors that acts as a dominant negative regulator of gene transcription. Four different isoforms of ICER (I, Igamma, II and IIgamma) are transcribed from the P2 promoter of the Crem gene. We previously found that each of the ICER isoforms is induced by parathyroid hormone in osteoblasts. The goal of the present study was to assess the function of ICER in bone by overexpressing ICER in osteoblasts of transgenic mice. ICER I and ICER II cDNAs, each containing an N-terminal FLAG epitope tag, were cloned downstream of a fragment containing 3.6 kb of the rat Col1a1 promoter and most of the rat Col1a1 first intron to produce pOBCol3.6-ICER I and pOBCol3.6-ICER II transgenes, respectively. Multiple lines of mice were generated bearing the ICER I and ICER II transgenes. At 8 weeks of age, ICER I and ICER II transgenic mice had lower body weights and decreased bone mineral density of femurs and vertebrae. Further studies were done with ICER I transgenic mice, which had greatly reduced trabecular bone volume and a markedly decreased bone formation rate in femurs. Osteoblast differentiation and osteocalcin expression were reduced in ex vivo bone marrow cultures from ICER I transgenic mice. ICER I antagonized the activity of ATF4 at its consensus DNA binding site in the osteocalcin promoter in vitro. Thus, transgenic mice with osteoblast-targeted overexpression of ICER exhibited osteopenia caused primarily by reduced bone formation. We speculate that ICER regulates the activity and/or expression of ATF/CREB factors required for normal bone formation.

The in vitro response of human osteoblasts to polyetheretherketone (PEEK) substrates compared to commercially pure titanium.

Polyetheretherketone (PEEK) is used as an alternative to titanium in medical devices. Previous in vitro studies examining PEEK have differed in their choice of polymer variant [PEEK or carbon-fiber reinforced PEEK (CFR-PEEK)], source of polymer (some of which are no longer available or for implantation) and cell type. While all studies demonstrated favorable cytocompatibility of the PEEK material, no studies are available which reflect the current state of the art of the material. Here, we use different forms of the only implantable grade PEEK available. These are compared with commercially pure titanium (cpTi) Grade 1 using a human primary osteoblast model. Sample materials were presented as industrially relevant surfaces. Machined or injection molded PEEK and CFR-PEEK were evaluated along with polished (Ra=0.200microm) and rough (Ra=0.554microm) cpTi. Osteoblast adhesion at 4h on injection molded variants of PEEK (Ra=0.095microm) and CFR-PEEK (Ra=0.350microm) material was comparable to titanium. Machined variants of PEEK (Ra=0.902microm) and CFR-PEEK (Ra=1.106microm) materials were significantly less. Proliferation at 48h determined by [(3)H]-thymidine incorporation was the greatest on the smoothest of all materials, the injection molded unfilled PEEK, which was significantly higher than the rough titanium control. The machined unfilled PEEK had the lowest DNA synthesis. RT-PCR for alkaline phosphatase, Type I collagen and osteocalcin normalized to glyceraldehyde-3-phosphate dehydrogenase revealed different patterns of mRNA levels. High mRNA levels for Type I collagen showed that CFR-PEEK stimulated osteoblast differentiation, whilst injection molded unfilled PEEK was less differentiated. Machined unfilled PEEK had comparable message levels of bone matrix proteins as rough titanium. All material variants permitted a degree of mineralization. Scanning electron microscopy at 3 days and 2 weeks in differentiation medium showed that human osteoblasts were well spread on all the different substrates. The varied response reported here at different time points during the study suggests that material formulation (unfilled PEEK or CFR-PEEK), subjection to industrial processing, surface roughness and topography may all influence the cellular response of osteoblasts to PEEK. Thus, differences in human osteoblast responses were found to the various samples of PEEK, but implantable grade PEEK, in general, was comparable in vitro to the bone forming capacity of rough titanium.

Therapeutic touch stimulates the proliferation of human cells in culture.

OBJECTIVES: Our objective was to assess the effect of Therapeutic Touch (TT) on the proliferation of normal human cells in culture compared to sham and no treatment. Several proliferation techniques were used to confirm the results, and the effect of multiple 10-minute TT treatments was studied. DESIGN: Fibroblasts, tendon cells (tenocytes), and bone cells (osteoblasts) were treated with TT, sham, or untreated for 2 weeks, and then assessed for [(3)H]-thymidine incorporation into the DNA, and immunocytochemical staining for proliferating cell nuclear antigen (PCNA). The number of PCNA-stained cells was also quantified. For 1 and 2 weeks, varying numbers of 10-minute TT treatments were administered to each cell type to determine whether there was a dose-dependent effect. RESULTS: TT administered twice a week for 2 weeks significantly stimulated proliferation of fibroblasts, tenocytes, and osteoblasts in culture (p = 0.04, 0.01, and 0.01, respectively) compared to untreated control. These data were confirmed by PCNA immunocytochemistry. In the same experiments, sham healer treatment was not significantly different from the untreated cultures in any group, and was significantly less than TT treatment in fibroblast and tenocyte cultures. In 1-week studies involving the administration of multiple 10-minute TT treatments, four and five applications significantly increased [(3)H]-thymidine incorporation in fibroblasts and tenocytes, respectively, but not in osteoblasts. With different doses of TT for 2 weeks, two 10-minute TT treatments per week significantly stimulated proliferation in all cell types. Osteoblasts also responded to four treatments per week with a significant increase in proliferation. Additional TT treatments (five per week for 2 weeks) were not effective in eliciting increased proliferation compared to control in any cell type. CONCLUSIONS: A specific pattern of TT treatment produced a significant increase in proliferation of fibro-blasts, osteoblasts, and tenocytes in culture. Therefore, TT may affect normal cells by stimulating cell proliferation.

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.

CREM deficiency in mice alters the response of bone to intermittent parathyroid hormone treatment.

CREM belongs to the ATF/CREB family of basic leucine zipper transcription factors. We previously showed that PTH induces ICER (inducible cAMP early repressor) in osteoblasts. ICER proteins, which are transcribed from the P2 promoter of the Crem gene, act as transcriptional attenuators. The objective of this study was to determine whether the Crem gene plays a role in the response of bone to intermittent PTH. Adult Crem knockout (KO) and wild type (WT) male mice were given daily subcutaneous injections of vehicle or hPTH(1-34) (160 mug/kg) for 10 days. Bone mineral content and density (BMC and BMD, respectively) were measured in femur and tibia by dual energy X-ray absorptiometry (DEXA). Bone morphometry was analyzed by X-ray computed microtomography (microCT) and histomorphometry. Serum bone turnover markers were measured. In vitro osteoclast formation assays were performed in bone marrow cultures treated with PTH or the combination of RANKL and M-CSF. KO mice had slightly higher basal bone mass than wild type mice. PTH treatment increased tibial BMC and BMD to a greater extent in WT mice compared to KO mice. PTH increased both cortical area and trabecular bone area in WT but not in KO femurs. PTH increased the bone formation rate and percent osteoblast surface to the same extent in femurs of WT and KO mice but increased osteoclast parameters and calvarial porosity to a greater extent in KO mice. PTH increased serum osteocalcin levels to the same extent in WT and KO mice. PTH-induced osteoclast formation was 2-fold greater in bone marrow cultures from KO mice. Collectively, our data suggest that the CREM deficiency in mice alters the response of bone to intermittent PTH treatment such that osteoclastogenesis is increased. Crem gene may specify the anabolic response to intermittent PTH treatment by restraining PTH-induced osteoclastogenesis.

Tendon and bone responses to a collagen-coated suture material.

Tendon to bone integration after rotator cuff repair is not a reproducible process. During repair, bioabsorbable and nonabsorbable suture material is universally used to facilitate the procedure. Improving the biological architecture of inert suture might aid in overall tendon to bone healing. The objective of our study is to enhance the bone to tendon union by absorbing type I collagen onto high strength nonabsorbable polyester/polyethylene suture commonly used in rotator cuff surgery. Our purpose was to evaluate the tendon and bone cellular response to this novel coated suture compared to uncoated suture. Primary human osteoblasts (HOBs) and tenocytes were plated onto polyester/polyethylene suture that was either uncoated or coated with type I bovine collagen. Cell adhesion to the sutures was assayed at 24 hours. Proliferation was determined at 48 hours by measuring [3H]- Thymidine incorporation in cells attached to the sutures. At 24 and 48 hours, respectively, cells grown on the collagen-coated suture showed a significantly greater response measured by adhesion and proliferation than cells grown on uncoated suture. At five days of culture, alkaline phosphatase activity and protein synthesis was significantly greater on the collagen-coated suture compared to uncoated. Collagen-coated polyester/polyethylene suture appears to stimulate adhesion, proliferation alkaline phosphatase, and protein synthesis more than uncoated sutures, and therefore may aid in the tendon to bone incorporation process critical to rotator cuff repair.

* 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.

Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss.

UNLABELLED: IL-7 is produced by stromal cells in bone marrow and is a major regulator of B and T lymphopoiesis. It is also a direct inhibitor of osteoclastogenesis in vitro. In this study we show that IL-7-deficient mice have increased OC and decreased trabecular bone volume compared with WT mice but mimic WT mice in the amount of trabecular but not cortical bone lost after ovariectomy. INTRODUCTION: Interleukin (IL)-7 is a potent regulator of lymphocyte development, which has significant effects on bone. Bone marrow cell cultures from IL-7 deficient (IL-7KO) mice produced significantly more TRACP(+) osteoclasts (OCs) than did cells from wildtype (WT) mice. A previous study found that treatment of mice with a neutralizing antibody to IL-7 blocked ovariectomy (OVX)-induced bone loss. We examined if differences exist between the bones of WT and IL-7KO mice and if OVX altered bone mass in IL-7KO mice. MATERIALS AND METHODS: Studies were in 2-month-old sham-operated (SHAM) and OVX female mice that were killed 4 weeks after surgery. IL-7KO mice and WT controls were in a C57BL/6 background. Both vertebrae (L(1)) and femora were evaluated by DXA, muCT, and histomorphometry. IL-7KO mice were confirmed as IL-7 deficient by their almost total lack of mature B cells in their bone marrow. RESULTS: There was significantly less trabecular bone volume in the vertebrae of IL-7KO mice than in WT mice. In addition, IL-7KO mice had significantly decreased (p < 0.05) trabecular number (13%) and increased trabecular spacing (15%). OVX decreased vertebral trabecular bone volume (TBV) by 21% (p < 0.05) in WT mice and by 22% (p < 0.05) in IL-7KO mice compared with SHAM. IL-7KO SHAM mice also had significantly less (30%) TBV (TA/TTA) in their femurs, as measured histomorphometrically, than did WT SHAM mice. Femurs from IL-7KO SHAM mice had significantly increased percent OC surface (23%) compared with WT SHAM. As in the vertebrae, OVX significantly decreased femoral TBV in both WT and IL-7KO mice by similar amounts (47% and 48%, respectively, p < 0.05 for both) compared with SHAM. However, OVX decreased cortical bone mass in WT but not in IL-7KO bones. We also examined bone marrow cells from WT and IL-7KO mice. Bone marrow cells from IL-7KO animals showed a significant increase in the number of TRACP(+) osteoclast-like cells (OCLs), which formed in cultures that were stimulated with macrophage-colony stimulating factor (M-CSF) and RANKL (both at 30 ng/ml). However, there was no significant difference in the number of OCLs that formed in B lymphocyte-depleted (B220(-)) bone marrow cell cultures from WT and IL-7KO mice. CONCLUSIONS: IL-7 deficiency in mice caused increased OC number in bone and decreased bone mass. OVX-induced bone loss in IL-7-deficient mice was selective and occurred in trabecular but not cortical bone.

T lymphocyte-deficient mice lose trabecular bone mass with ovariectomy.

UNLABELLED: We examined OVX-induced bone loss in three TLD mouse models. In TLD mice, OVX caused trabecular bone loss equivalent to that of WT. In contrast, cortical bone loss with OVX was variable. We conclude that T lymphocytes do not influence OVX-induced trabecular bone loss. INTRODUCTION: We examined ovariectomy (OVX)-induced bone loss in three T lymphocyte-deficient (TLD) mouse models: nude mice, recombination activating gene 2-deficient (RAG2 KO) mice, and T cell receptor alpha chain-deficient (TCRalpha KO) mice. MATERIALS AND METHODS: Bone mass was examined by DXA, microCT, and histomorphometry. We also examined the effect of OVX on T lymphocytes in the bone marrow and spleens of wildtype (WT) mice and on in vitro osteoclastogenesis and colony forming unit-granulocyte macrophage (CFU-GM) activity in the bone marrow of WT and nude mice. RESULTS: In WT mice, OVX did not alter T lymphocyte number in the bone marrow but did increase T lymphocytes in the spleen. Comparison of bone mass in nude, RAG2 KO, and TCRalpha KO mice with WT as measured by DXA showed decreased femoral bone mass in nude mice and increased vertebral bone mass in RAG2 KO mice. In TCRalpha KO mice, femoral, tibial, and vertebral bone mass were decreased. In vertebrae and long bones, bone loss with OVX was consistently present in WT mice but variably present in TLD mice as measured by DXA. In contrast, microCT and histomorphometry showed similar trabecular bone loss after OVX in all mice. However, femoral cortical bone loss occurred only in WT and RAG2 KO mice. OVX produced similar trabecular bone loss in WT and TCRalpha KO mice and also induced cortical bone loss in both. Histomorphometry showed that TRACP(+) area in bones was increased by OVX in femurs from both WT and nude mice as was in vitro osteoclast-like cell formation and CFU-GM activity. CONCLUSIONS: These results show that OVX caused similar trabecular bone loss in both WT and TLD mice. The ability of DXA and measurement of cortical bone loss to show OVX-induced effects on bone mass was variable. It seems that T lymphocytes are not critical for OVX-induced trabecular bone loss in these mouse models.

Transgenic mice with osteoblast-targeted insulin-like growth factor-I show increased bone remodeling.

To determine the effects of locally-expressed insulin-like growth factor (IGF-I) on bone remodeling, a transgene was produced in which murine IGF-I cDNA was cloned downstream of a gene fragment comprising 3.6 kb of 5' upstream regulatory sequence and most of the first intron of the rat Col1a1 gene. The construct was expressed at the mRNA and protein level in transfected osteoblasts. Five lines of transgenic mice were generated by embryo microinjection. Transgene mRNA levels were highest in calvaria, long bone and tendon, and lower in skin. Serum IGF-I and body weight were increased in males and females only in the highest expressing line. Histomorphometry showed that transgenic calvaria were wider and had greater marrow area and bone area. Transgenic calvaria had increased osteoclast number per bone surface. Percent collagen synthesis and cell replication were increased in transgenic calvaria. Femur length, cortical width and cross-sectional area were increased in transgenic femurs of the highest expressing line, while femoral trabecular bone volume was little affected. Thus, broad overexpression of IGF-I in cells of the osteoblast lineage increased indices of bone formation and resorption.

Matrix-mediated retention of in vitro osteogenic differentiation potential and in vivo bone-forming capacity by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion.

Mesenchymal stem cells (MSCs) represent an attractive cell source for tissue engineering applications, since they are readily isolated from adult bone marrow and have the ability to differentiate along multiple mesenchymal lineages, including osteogenic. Currently, utilization of MSCs for bone tissue engineering is limited because of the attenuation of their osteogenic differentiation potential and in vivo bone-forming capacity following ex vivo expansion on conventional tissue culture plastic (TCP). Previously, we demonstrated that a denatured type I collagen (DC) matrix promotes the maintenance of MSC in vitro osteogenic differentiation potential during ex vivo expansion in contrast to TCP. In this study, we further demonstrate that the maintenance of MSC osteogenic differentiation potential is primarily due to the ability of DC matrix to influence the retention of early passage osteogenic functions in late passage (LP) cells during ex vivo expansion, in contrast to solely enhancing attenuated LP cellular functions during osteogenic differentiation. Serum-associated factors played a significant role in influencing the retention of MSC osteogenic differentiation potential during expansion on the DC matrix. Significantly, the results show that although LP cells expanded ex vivo on TCP highly attentuate their in vivo bone-forming capacity, the expansion of MSCs on DC matrix preserves this ability as determined by histological, histomorphometric, and bone mineral density evaluations of MSC-seeded hydroxyapatite/tricalcium phosphate scaffolds following an 8-week implantation period within a heterotopic muscle pouch model. These findings provide further insight into the importance of matrix-mediated effects on MSC function and selective factors important in this process.

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.

Effect of osteoblast-targeted expression of bcl-2 in bone: differential response in male and female mice.

UNLABELLED: Transgenic mice (Col2.3Bcl-2) with osteoblast-targeted human Bcl-2 expression were established. Phenotypically, these mice were smaller than their wildtype littermates and showed differential effects of the transgene on bone parameters and osteoblast activity dependent on sex. The net effect was an abrogation of sex differences normally observed in wildtype mice and an inhibition of bone loss with age. Ex vivo osteoblast cultures showed that the transgene had no effect on osteoblast proliferation, but decreased bone formation. Estrogen was shown to stimulate endogenous Bcl-2 message levels. These studies suggest a link between Bcl-2 and sex regulation of bone development and age-related bone loss. INTRODUCTION: Whereas Bcl-2 has been shown to be an important regulator of apoptosis in development, differentiation, and disease, its role in bone homeostasis and development is not well understood. We have previously showed that the induction of glucocorticoid-induced apoptosis occurred through a dose-dependent decrease in Bcl-2. Estrogen prevented glucocorticoid-induced osteoblast apoptosis in vivo and in vitro by preventing the decrease in Bcl-2 in osteoblasts. Therefore, Bcl-2 may be an important regulator of bone growth through mechanisms that control osteoblast longevity and function. MATERIALS AND METHODS: Col2.3Bcl-2 mice were developed carrying a 2.3-kb region of the type I collagen promoter driving 1.8 kb of human Bcl-2 (hBcl-2). Tissue specific expression of hBcl-2 in immunoassays validated the transgenic animal model. Histomorphometry and DXA were performed. Proliferation, mineralization, and glucocorticoid-induced apoptosis were examined in ex vivo cultures of osteoblasts. The effect of estrogen on mouse Bcl-2 in ex vivo osteoblast cultures was assayed by RT-PCR and Q-PCR. RESULTS AND CONCLUSIONS: Two Col2.3Bcl-2 (tg/+) founder lines were established and appeared normal except that they were smaller than their nontransgenic wildtype (+/+) littermates at 1, 2, and 6 months of age, with the greatest differences at 2 months. Immunohistochemistry showed hBcl-2 in osteoblasts at the growth plate and cortical surfaces. Nontransgenic littermates were negative. Western blots revealed hBcl-2 only in type I collagen-expressing tissues. Histomorphometry of 2-month-old mice showed a significant decrease in tg/+ calvaria width with no significant differences in femoral trabecular area or cortical width compared with +/+. However, tg/+ males had significantly more trabecular bone than tg/+ females. Female +/+ mice showed increased bone turnover with elevated osteoblast and osteoclast parameters compared with +/+ males. Col2.3Bcl-2 mice did not show such significant differences between sexes. Male tg/+ mice had a 76.5 +/- 1.5% increase in ObS/BS with no significant differences in bone formation rate (BFR) or mineral apposition rate (MAR) compared with male +/+ mice. Transgenic females had a significant 48.4 +/- 0.1% and 20.1 +/- 5.8% decrease in BFR and MAR, respectively, compared with +/+ females. Osteoclast and osteocyte parameters were unchanged. By 6 months, femurs from female and male +/+ mice had lost a significant amount of their percent of trabecular bone compared with 2-month-old mice. There was little to no change in femoral bone in the tg/+ mice with age. Ex vivo cultures of osteoblasts from +/+ and Col2.3Bcl-2 mice showed a decrease in mineralization, no effect on proliferation, and an inhibition of glucocorticoid-induced apoptosis in Col2.3Bcl-2 cultures. Estrogen was shown to increase mouse Bcl-2 transcript levels in osteoblast cultures of wildtype mice, supporting a role for Bcl-2 in the sex-related differences in bone phenotype regulated by estrogen. Therefore, Bcl-2 differentially affected bone phenotype in male and female transgenic mice, altered bone cell activity associated with sex-related differences, and decreased bone formation, suggesting that apoptosis is necessary for mineralization. In addition, Bcl-2 targeted to mature osteoblasts seemed to delay bone development, producing a smaller transgenic mouse compared with wildtype littermates. These studies suggest that expression of Bcl-2 in osteoblasts is important in regulating bone mass in development and in the normal aging process of bone.

Do cyclooxygenase-2 knockout mice have primary hyperparathyroidism?

The absence of cyclooxygenase-2 (COX-2) activity in vitro reduces differentiation of both bone-forming and bone-resorbing cells. To examine the balance of COX-2 effects on bone in vivo, we studied COX-2 knockout (KO) and wild-type (WT) mice. After weaning, KO mice died 4 times faster than WT mice, consistent with reports of progressive renal failure in KO mice. Among KO mice killed at 4 months of age, some had renal failure with marked secondary hyperparathyroidism, but others appeared healthy. On the assumption that renal failure was not inevitable in COX-2 KO mice and that phenotypic differences might increase with age, we studied KO mice surviving to 10 months of age with serum creatinine levels similar to those of WT mice. In 10-month-old male KO mice, serum calcium and PTH, but not phosphorus, levels were increased compared with those in WT mice. 1,25-Dihydroxyvitamin D(3) levels were markedly elevated in KO mice. Skeletal analysis showed small nonsignificant decreases in cortical bone density by BMD and either an increase (distal femur, by microcomputed tomography) or no difference (distal femur, by static histomorphometry) in trabecular bone density in KO mice. There was a trend toward increased percent osteoblastic and osteoclastic surfaces, and on dynamic histomorphometry, the rates of trabecular bone formation and mineral apposition were increased in KO mice relative to WT mice. Similar trends were observed for most parameters in 10-month-old female COX-2 KO mice. However, rates of trabecular bone formation and mineral apposition were increased in 10-month-old WT females compared with males and did not increase further in female KO mice. These data suggest that COX-2 KO mice with intact renal function have primary hyperparathyroidism, and that effects of increased PTH and 1,25-dihydroxyvitamin D(3) to increase bone turnover may compensate for the absence of COX-2.

The inflammatory responses to silk films in vitro and in vivo.

Silks have a long history of biomedical use as sutures. Silk can be purified, chemically modified to attach RGD sequences and processed into highly porous scaffolds for tissue engineering. We report biocompatibility studies of silk films (with or without covalently bound RGD) that were seeded with bone-marrow derived mesenchymal stem cells (MSC) and (a) cultured in vitro with human MSC or (b) seeded with autologous rat MSC and implanted in vivo. Controls for in vitro studies included tissue culture plastic (TCP; negative control), TCP with lipopolysaccharide (LPS) in the cell culture medium (positive control), and collagen films; controls for in vivo studies included collagen, PLA and TCP. After 9 h of culture, the expression of the pro-inflammatory Interleukin 1 beta (IL-1beta) and inflammatory cyclooxygenase 2 (COX-2) in human MSC were comparable for silk, collagen and TCP. After 30 and 96 h, gene expression of IL-1beta and COX-2 in MSC returned to the baseline (pre-seeding) levels. These data were corroborated by measuring IL-1beta and prostaglandin E2 levels in culture medium. The rate of cell proliferation was higher on silk films than either on collagen or TCP. In vivo, films made of silk, collagen or PLA were seeded with rat MSCs, implanted intramuscularly in rats and harvested after 6 weeks. Histological and immunohistochemical evaluation of silk explants revealed the presence of circumferentially oriented fibroblasts, few blood vessels, macrophages at the implant-host interface, and the absence of giant cells. Inflammatory tissue reaction was more conspicuous around collagen films and even more around PLA films when compared to silk. These data suggest that (a) purified degradable silk is biocompatible and (b) the in vitro cell culture model (hMSC seeded and cultured on biomaterial films) gave inflammatory responses that were comparable to those observed in vivo.

Transforming growth factor-beta 1 (TGF-beta1) prevents the age-dependent decrease in bone formation in human osteoblast/implant cultures.

Titanium implants have been extensively used in orthopedic surgery and dentistry. Most of the patients who receive such implants are elderly with a compromised ability to heal and form new bone. By using an in vitro osteoblast/implant culture system, the potency of TGF-beta1 in enhancing mineralization of human osteoblast cultures from elderly subjects was investigated in this study. Primary human osteoblast (HOB) cells obtained from different age group human subjects [Young (Y), Middle (M), and Old (O)] were cultured on Ti alloy (Ti-6Al-4V) disks with or without continuous administration of 0.2 ng/mL TGF-beta1 in the medium for 2 or 4 weeks. TGF-beta1 significantly (p < 0.05) increased calcium content and the size of calcified nodules on implant disks in the O group, but had no effect on the Y or M groups. The number of calcified nodules was not different with or without TGF-beta1 in all age groups. As measured by Northern blot analysis and RT-PCR, TGF-beta1 significantly increased the expression of bone-specific extracellular matrix proteins, including alkaline phosphatase, Type I collagen, bone sialoprotein and osteocalcin, after both 2 and 4 weeks in the O group but not in the Y group. In conclusion, TGF-beta1 enhances mineralization on implant materials of osteoblast cultures from elderly human subjects.