The Role of Research Experiences in the Training of Oral and Maxillofacial Surgeons.

The importance of active adult learning methods and critical thinking skills is appreciated in dental and OMFS residency training. Known barriers to research are finding time in the curriculum and funding needed for research experiences. These barriers have inspired many institutions to design programs to provide research opportunities, but they can be expensive and of minimal interest to those not planning academic careers. During OMFS residency training, the primary emphasis is on mastery of all aspects of surgical care. Strong partnerships between PhD researchers and OMFS clinical investigators, formed to advance the field, can also have an impact on trainees' involvement in research and their understanding of rigorous evidence-based principles of clinical care.

Synergistic stimulation of osteoblast differentiation of rat mesenchymal stem cells by leptin and 25(OH)D3 is mediated by inhibition of chaperone-mediated autophagy.

BACKGROUND: Vitamin D is important for the mineralization of bones by stimulating osteoblast differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs are a target of vitamin D action, and the metabolism of 25(OH)D3 to biologically active 1α,25(OH)2D3 in BMMSCs promotes osteoblastogenesis in an autocrine/paracrine manner. Our previous study with human BMMSCs showed that megalin is required for the 25(OH)D3-DBP complex to enter cells and for 25(OH)D3 to stimulate osteoblast differentiation in BMMSCs. Furthermore, we reported that leptin up-regulates megalin in those cells. Leptin is a known inhibitor of PI3K/AKT-dependent chaperone-mediated autophagy (CMA). In this study, we tested the hypothesis that leptin acts synergistically with 25(OH)D3 to promote osteoblastogenesis in rat BMMSCs by a mechanism that entails inhibition of PI3K/AKT-dependent CMA. METHODS: BMMSCs were isolated from rat bone marrow (4-week-old male SD rats); qRT-PCR and western immunoblots or immunofluorescence were used to evaluate the expression of megalin, ALP, COL1A1, RUNX2, OSX, OSP, and CMA in rBMMSCs. The osteoblast differentiation was evaluated by ALP activity, ALP staining, and calcium deposition. The viability of rBMMSCs was assessed with the CCK-8 kit. Biosynthesis of 1α,25(OH)2D3 was measured by a Rat 1α,25(OH)2D3 ELISA Kit. RESULTS: The combination of leptin and 25(OH)D3 treatment significantly enhanced osteoblast differentiation as shown by ALP activity, ALP staining, and calcium deposition, the expression of osteogenic genes ALP, COL1A1, RUNX2, OSX, and OSP by qRT-PCR and western immunoblots in rBMMSCs. Leptin enhanced the expression of megalin and synthesis of 1α,25(OH)2D3 in rBMMSCs. Our data showed that leptin inhibited CMA activity of rBMMSCs by activating PI3K/AKT signal pathway; the ability of leptin to enhance 25(OH)D3 promoted osteoblast differentiation of rBMMSCs was weakened by the PI3K/AKT signal pathway inhibitor. CONCLUSIONS: Our data reveal the mechanism by which leptin and 25(OH)D3 promote osteoblast differentiation in rBMMSCs. Leptin promoted the expression of megalin by inhibiting CMA, increased the utilization of 25(OH)D3 by rBMMSCs, and enhanced the ability of 25(OH)D3 to induce osteoblast differentiation of rBMMSCs. PI3K/AKT is at least partially involved in the regulation of CMA. These data indicate the importance of megalin in BMMSCs for vitamin D's role in skeletal health.

Combined injury: irradiation with skin or bone wounds in rodent models.

A radiation combined injury is defined as an injury that occurs in the setting of irradiation, such as those expected after a nuclear accident, radiation dispersal device release (a 'dirty bomb'), or a nuclear weapon detonation. There is much research on irradiation-associated burns and their healing, but there is less known about other injuries sustained in the context of irradiation. Animal models are limited in their correlations to clinical situations but can support research on specific questions about injuries and their healing. Mouse models of irradiation with skin or bone wounds are validated as highly reproducible and quantitative. They show dose-dependent impairment of wound healing, with later recovery. Irradiation-induced delay of bone wound healing was mitigated to different extents by single doses of gramicidin S-nitroxide JP4-039, a plasmid expressing manganese superoxide dismutase, amifostine/WR2721, or the bifunctional sulfoxide MMS-350. These models should be useful for research on mechanisms of radiation dermal and osseous damage and for further development of new radioprotectors. They also provide information of potential relevance to the effects of clinical radiation therapies.

Fibroblast growth factor 23 counters vitamin D metabolism and action in human mesenchymal stem cells.

Chronic kidney disease (CKD) is associated with elevated circulating fibroblast growth factor 23 (FGF23), impaired renal biosynthesis of 1α,25-dihydroxyvitamin D (1α,25(OH)2D), low bone mass, and increased fracture risk. Our previous data with human mesenchymal stem cells (hMSCs) indicated that vitamin D metabolism in hMSCs is regulated as it is in the kidney and promotes osteoblastogenesis in an autocrine/paracrine manner. In this study, we tested the hypothesis that FGF23 inhibits vitamin D metabolism and action in hMSCs. hMSCs were isolated from discarded marrow during hip arthroplasty, including two subjects receiving hemodialysis and a series of 20 subjects (aged 49-83 years) with estimated glomerular filtration rate (eGFR) data. The direct in vitro effects of rhFGF23 on hMSCs were analyzed by RT-PCR, Western immunoblot, and biochemical assays. Ex vivo analyses showed positive correlations for both secreted and membrane-bound αKlotho gene expression in hMSCs with eGFR of the subjects from whom hMSCs were isolated. There was downregulated constitutive expression of αKlotho, but not FGFR1 in hMSCs obtained from two hemodialysis subjects. In vitro, rhFGF23 countered vitamin D-stimulated osteoblast differentiation of hMSCs by reducing the vitamin D receptor, CYP27B1/1α-hydroxylase, biosynthesis of 1α,25(OH)2D3, and signaling through BMP-7. These data demonstrate that dysregulated vitamin D metabolism in hMSCs may contribute to impaired osteoblastogenesis and altered bone and mineral metabolism in CKD subjects due to elevated FGF23. This supports the importance of intracellular vitamin D metabolism in autocrine/paracrine regulation of osteoblast differentiation in hMSCs.

Obesity and leptin influence vitamin D metabolism and action in human marrow stromal cells.

Obesity is associated with low serum 25-hydroxyvitamin D [s25(OH)D], high serum leptin, and generally high bone mineral density (BMD). Human Marrow Stromal Cells (hMSCs) differentiate to osteoblasts and are both a target and source of vitamin D metabolites in bone marrow. There is no information about the influence of obesity on vitamin D metabolism and osteoblastogenesis in hMSCs and little about direct effects of leptin on hMSCs. In this study, we tested the hypotheses that 1) obesity has an influence on the ex vivo constitutive expression of vitamin D-hydroxylase genes in hMSCs, and 2) recombinant human (rh) Leptin regulates the D-hydroxylases and promotes osteoblastogenesis in hMSCs. In a cohort of female subjects undergoing joint replacement surgery, the effects of Body Mass Index (BMI) and Fat Mass Index (FMI) on BMD T-scores and s25(OH)D were evaluated. hMSCs were isolated from bone tissues discarded during surgery. The direct effects of rh-Leptin on osteoblast differentiation and D-related genes in hMSCs were examined in vitro. There were positive correlations for BMD T-score of femoral neck and spine with BMI and FMI. Serum 25(OH)D levels in obese subjects were 71% of that in non-obese counterparts (p = 0.001). hMSCs from obese women had higher constitutive expression of CYP27A1/25-hydroxylase and vitamin D receptor. Those findings raised the mechanistic question of how obesity could influence vitamin D metabolism and osteoblast differentiation in hMSCs. Treating hMSCs with rh-Leptin in vitro significantly stimulated osteoblastogenesis. In addition, leptin downregulated CYP24A1 and upregulated CYP27B1, CYP27A1 and VDR, which play vital roles in vitamin D metabolism. Furthermore, co-treatment with leptin and vitamin D3 metabolites promoted ALP activity compared with either alone. This research demonstrates links between obesity, vitamin D metabolism, and osteoblastogenesis by which leptin's direct effects on D-metabolism and osteoblast differentiation in hMSCs may protect bone from low s25(OH)D in obese subjects.

Megalin mediates 25-hydroxyvitamin D3 actions in human mesenchymal stem cells.

Osteoblast differentiation of human mesenchymal stem cells (hMSCs) is stimulated by 1α,25-dihydroxycholecalciferol [1α,25(OH)2D3] and 25-hydroxycholecalciferol [25(OH)D3]; the latter's effects require intracellular hydroxylation to 1α,25(OH)2D3. Thus, hMSCs are both a source of and target for 1α,25(OH)2D3. Megalin is a transmembrane receptor for serum d-binding protein (DBP) in kidney cells and is required for uptake of the 25(OH)D3-DBP complex. We tested the hypothesis that megalin is required for D actions in hMSCs with cells from surgically discarded marrow for RT-PCR, for effects of 25(OH)D3 and 1α,25(OH)2D3, for 1α,25(OH)2D3 biosynthesis, for osteoblastogenesis, and for small interfering RNA for megalin (si-Meg) and control (si-Ctr). In hMSCs with high constitutive megalin expression, both 1α,25(OH)2D3 and 25(OH)D3 stimulated osteoblastogenesis (P < 0.05), but only 1α,25(OH)2D3 did so in hMSCs with lower megalin (lo-Meg, P < 0.001) or in si-Meg cells (P < 0.05). In addition, 1α,25(OH)2D3 biosynthesis was significantly lower in lo-Meg (46%, P = 0.034) and in si-Meg (23%, P < 0.001) than each control. Leptin significantly stimulated megalin expression 2.1-fold in lo-Meg cells (P < 0.01). These studies show that megalin is expressed in hMSCs and is required for the biosynthesis of 1α,25(OH)2D3 and for the 25(OH)D3/DBP complex to stimulate vitamin D receptor targets and osteoblastogenesis.-Gao, Y., Zhou, S., Luu, S., Glowacki, J. Megalin mediates 25-hydroxyvitamin D3 actions in human mesenchymal stem cells.

Dehydroepiandrosterone and Bone.

In humans, dehydroepiandrosterone (DHEA), secreted mainly from the adrenal cortex, and its sulfate ester, DHEAS, are the most abundant circulating steroids. DHEA/DHEAS possess pleiotropic effects in human aging, bone, metabolic diseases, neurologic function/neurodegenerative diseases, cancer, immune system and disorders, cardiovascular diseases, diabetes, muscle function, sexual dysfunction, and other health conditions. The age-related reduced levels of DHEA and DHEAS are associated with bone mineral density measures of osteopenia and osteoporosis. Clinical, epidemiological, and experimental studies indicate that DHEA replacement therapy may be beneficial for bone health through its inhibition of skeletal catabolic IL-6 and stimulation of osteoanabolic IGF-I-mediated mechanisms. Studies with primary cultures of human bone marrow-derived mesenchymal stem cells (hMSCs) were used to show that DHEA stimulates osteoblastogenesis. The in vitro stimulation of both osteoblastogenesis and IGF-I gene expression by DHEA in hMSCs requires IGF-I receptor, PI3K, p38 MAPK, or p42/44 MAPK signaling pathways. The in vitro inhibition of IL-6 secretion in hMSCs by DHEA was more consistent and extensive than by estradiol or dihydrotestosterone. In summary, evidence from us and others indicates that DHEA may be useful for treating bone diseases through its inhibition of skeletal catabolic IL-6 and stimulation of anabolic IGF-I-mediated mechanisms.

Development of tensile strength methodology for murine skin wound healing.

In this study, a methodology was evaluated and improved to quickly measure the tensile strength of murine skin in a biomechanical assay for an incisional wound healing model. The aim was to streamline and enhance the wound model, skin specimen preparation, and tensile test so that large numbers of fresh tissue could be tested reliably and rapidly. Linear incisions of 25-mm length were made in the dorsal skin of mice along the spine and metallic staples were used to close the wound. After 20 days, the mice were sacrificed, and a square-shaped section of skin containing the linear incision was excised. Two metallic punches were fabricated and used to punch 15-mm long strips of skin of 2 mm width whose length was orthogonal to the direction of incision. The tensiometer configuration was modified to expedite tensile measurements on fresh skin, and load-to-failure was measured for each strip of skin from the cephalad to the caudal region. We evaluated sources of error in the animal model and the testing protocol and developed procedures to maximize speed and reproducibility in tensile strength measurements. This report provides guidance for efficient and reproducible tensile strength measurement of large numbers of skin specimens from freshly sacrificed animals. •Tattoo placement to identify the two ends of the healing incisional wound assisted in decreasing error in the position and orientation of tensile strips.•Custom-made punches to prepare skin strips for tensile testing helped conduct tensile tests of fresh tissue rapidly.•Alteration of the manual grips of the tensile tester enabled specimens to be gripped rapidly to significantly accelerate testing for each skin strip.

Synergistic effect of 1α,25-dihydroxyvitamin D3 and 17ß-estradiol on osteoblast differentiation of pediatric MSCs.

Vitamin D is essential for mineral homeostasis and contributes to bone metabolism by stimulating osteoblast differentiation of marrow stromal cells (MSCs). In this study, we used MSCs from pre-pubertal girls and boys to test the hypothesis that 1α,25(OH)2D and 17ß-estradiol have synergistic effects on these MSCs, and what mechanism is involved. With IRB approval, we isolated MSCs from discarded excess iliac marrow graft from children undergoing alveolar cleft repair. Plasma was available from 8 female (9.3±0.2years) and 8 male (9.6±0.1years) subjects for hormone assays [25(OH)D, total testosterone, 17ß-estradiol, estrone, DHEA-S, Growth Hormone, IGF-I]. RT-PCR was used for gene expression. Alkaline phosphatase (ALP) activity was used to measure osteoblast differentiation at day 7; alizarin red was used to measure matrix mineralization at day 21. All subjects were pre-pubertal based on their hormone levels. Serum 25(OH)D levels ranged from 13.1 to 26.4ng/mL, with 75% below 20ng/mL. Constitutive gene expression of VDR and ERα, ß varied from subject to subject with no association with sex or serum chemistries. In osteoblastogenic medium, 1α,25(OH)2D3 (10nM) increased ALP activity by 36% (p<0.05) in MSCs; 10nM of E2 was not stimulatory but the combination of 1α,25(OH)2D3 and E2 increased ALP 151% (p<0.05 vs. control) and by 84.5% (p<0.05 vs. 1α,25(OH)2D3 alone). The combination of 1α,25(OH)2D3 and E2 significantly increased mineralization 11-fold, compared with either agent alone. Twenty-four hour treatment with 1α,25(OH)2D3 (10nM) or E2 (10nM) upregulated each other's receptor by as much as 5.8-fold for ERα and 2.9-fold for the VDR. In summary, 1α,25(OH)2D3 stimulated osteoblast differentiation and matrix mineralization with MSCs from pre-pubertal subjects, with a synergistic effect of E2, mediated by upregulated receptor levels, at least in part. These studies add new information about the regulation of human osteoblast differentiation, effects of 1α,25(OH)2D3 and E2 on MSCs, and the importance of vitamin D for skeletal health.

Decoy TRAIL receptor CD264: a cell surface marker of cellular aging for human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are a mixture of progenitors that are heterogeneous in their regenerative potential. Development of MSC therapies with consistent efficacy is hindered by the absence of an immunophenotype of MSC heterogeneity. This study evaluates decoy TRAIL receptor CD264 as potentially the first surface marker to detect cellular aging in heterogeneous MSC cultures. METHODS: CD264 surface expression, regenerative potential, and metrics of cellular aging were assessed in vitro for marrow MSCs from 12 donors ages 20-60 years old. Male and female donors were age matched. Expression of CD264 was compared with that of p16, p21, and p53 during serial passage of MSCs. RESULTS: When CD264+ cell content was 20% to 35%, MSC cultures from young (ages 20-40 years) and older (ages 45-60 years) donors proliferated rapidly and differentiated extensively. Older donor MSCs containing < 35% CD264+ cells had a small size and negligible senescence despite the donor's advanced chronological age. Above the 35% threshold, CD264 expression inversely correlated with proliferation and differentiation potential. When CD264+ cell content was 75%, MSCs were enlarged and mostly senescent with severely compromised regenerative potential. There was no correlation of the older donors' chronological age to either CD264+ cell content or the regenerative potential of the donor MSCs. CD264 was upregulated after p53 and had a similar expression profile to that of p21 during serial passage of MSCs. No sex-linked differences were detected in this study. CONCLUSIONS: These results suggest that CD264 is a surface marker of cellular age for MSCs, not the chronological age of the MSC donor. CD264 is first upregulated in MSCs at an intermediate stage of cellular aging and remains upregulated as aging progresses towards senescence. The strong inverse correlation of CD264+ cell content to the regenerative potential of MSCs has possible application to assess the therapeutic potential of patient MSCs, standardize the composition and efficacy of MSC therapies, and facilitate aging research on MSCs.

Chronic kidney disease and vitamin D metabolism in human bone marrow-derived MSCs.

Vitamin D that is synthesized in the skin or is ingested undergoes sequential steps of metabolic activation via a cascade of cytochrome P450 enzymatic hydroxylations in the liver and kidney to produce 1α,25-dihydroxyvitamin D (1α,25(OH)2 D). There are many tissues that are able to synthesize 1α,25(OH)2 D, but the biological significance of extrarenal hydroxylases is unresolved. Human marrow-derived mesenchymal stem cells (marrow stromal cells, hMSCs) give rise to osteoblasts, and their differentiation is stimulated by 1α,25(OH)2 D. In addition to being targets of 1α,25(OH)2 D, hMSCs can synthesize it; on the basis of those observations, we further examined the local autocrine/paracrine role of vitamin D metabolism in osteoblast differentiation. Research with hMSCs from well-characterized subjects provides an innovative opportunity to evaluate the effects of clinical attributes on the regulation of hMSCs. Like the renal 1α-hydroxylase, the enzyme in hMSCs is constitutively decreased with age and chronic kidney disease (CKD); both are regulated by PTH1-34, insulin-like growth factor 1, calcium, 1α,25(OH)2 D, 25(OH)D, and fibroblast growth factor 23. CKD is associated with impaired renal biosynthesis of 1α,25(OH)2 D, low bone mass, and increased fracture risk. Studies with hMSCs from CKD patients or aged subjects indicate that circulating 25(OH)D may have an important role in osteoblast differentiation on vitamin D metabolism and action in hMSCs.

Glycinol enhances osteogenic differentiation and attenuates the effects of age on mesenchymal stem cells.

AIM: Phytoestrogens, such as glycinol, have recently gained significant attention as an alternative therapy for osteoporosis due to their structural similarity to estradiol and their bone-generating potential. METHODS: The osteogenic effects of glycinol were investigated in human bone marrow mesenchymal stem cells (BMSCs) derived from older (>50 years old) and younger subjects (<25 years old). RESULTS: BMSCs isolated from older donors demonstrated reduced osteogenesis. 17ß-estradiol and glycinol exposure rescued the age-related reduction in osteogenic differentiation of BMSCs. These results correlated with the induction of osteogenic genes and estrogen receptor-α (ER-α) following glycinol treatment. ER antagonist studies further support that glycinol promotes osteogenesis through ER signaling. CONCLUSION: The results from these studies support investigating glycinol as a potential preventive or treatment for osteoporosis.

Administration of an activin receptor IIB ligand trap protects male juvenile rhesus macaques from simian immunodeficiency virus-associated bone loss.

HIV-infected individuals are at an increased risk of osteoporosis despite effective viral suppression. Observations that myostatin null mice have increased bone mass led us to hypothesize that simian immunodeficiency virus (SIV)-associated bone loss may be attenuated by blocking myostatin/TGFß signaling. In this proof-of-concept study, pair-housed juvenile male rhesus macaques were inoculated with SIVmac239. Four weeks later, animals were treated with vehicle or Fc-conjugated soluble activin receptor IIB (ActR2B·Fc, iv. 10mg∗kg-1∗week-1) - an antagonist of myostatin and related members of TGFß superfamily. Limb and trunk bone mineral content (BMC) and density (BMD) using dual-energy X-Ray absorptiometry, circulating markers of bone growth and turnover, and serum testosterone levels were measured at baseline and during the 12-week intervention period. The increase in BMC was significantly greater in the ActRIIB.Fc-treated group (+8g) than in the placebo group (-4g) (p<0.05). BMD also increased significantly more in the ActRIIB.Fc-treated macaques (+0.03g/cm2) than in the placebo-treated animals (+0g/cm2) (p<0.005). Serum osteocalcin was about two-fold higher in the ActRIIB.Fc-treated group than in the placebo group (p<0.05), but serum C-terminal telopeptide and testosterone levels did not differ significantly between groups. The expression levels of TNFalpha (p<0.05), GADD45 (p<0.005), and sclerostin (p<0.038) in the bone-marrow were significantly lower in the ActRIIB.Fc-treated group than in the placebo group. CONCLUSION: The administration of ActRIIB.FC in SIV-infected juvenile macaques significantly increases BMC and BMD in association with reduced expression levels of markers of bone marrow inflammation.

Influence of osteoarthritis grade on molecular signature of human cartilage.

Articular chondrocytes maintain cartilage matrix turnover and have the capacity for anabolic and catabolic activities that can be influenced by injury and disease. This study tested the hypothesis that catabolic genes are upregulated with regional osteoarthritis (OA) disease severity within a joint. With IRB approval, specimens of knee cartilage obtained as discarded tissues from subjects undergoing arthroplasty were partitioned for each subject by OA disease severity and evaluated for gene expression by RT-PCR. There was regional OA grade-associated upregulation of expected inflammatory mediators TNF-α, TNF receptors, IFN-γ, and interleukins as well as genes encoding proteolytic enzymes, including Adamts-5 and MMPs. Osteoclast-related genes, cathepsin K, tartrate-resistant acid phosphatase (TRAP), RANKL, RANK, M-CSF, and c-fms, but not osteoprotegerin, were induced in advanced grades. In vitro treatment of normal human chondrocytes with interleukin-1ß upregulated similar genes; this provides evidence that chondrocytes per se can be the source of osteoclast-related factors. Immunohistochemical staining showed that RANK- and RANKL-positive cells were abundant in advanced grades, especially in chondrocyte clusters. This suggests a possible autocrine mechanism by which an osteoclast phenotype is induced in articular chondrocytes. In sum, these studies identified gene expression signatures in human OA cartilage based upon regional disease severity within a joint. There was an effect of OA Grade on expression of osteoclastic lytic enzymes and regulatory factors in human articular chondrocytes. Induction of an osteoclast-like phenotype in chondrocytes may be part of OA progression and suggests specific therapeutic approaches.

Parathyroid hormone and its receptor gene polymorphisms: implications in osteoporosis and in fracture healing.

Parathyroid glands secrete parathyroid hormone (PTH) which plays multiple roles in calcium homeostasis and in bone remodeling. Secretion of PTH is regulated by extracellular calcium levels and other humoral factors including 1α,25(OH)2D3. PTH regulates gene expression and induces biological effects directly and indirectly. The human gene encoding PTH is located on chromosome 11. In this review, we study the diverse PTH along with its receptor gene polymorphisms and their association with osteoporosis and fracture healing. Genetic factors are associated with osteoporosis by influencing bone mineral density (BMD), bone turnover, calcium homeostasis, and susceptibility to osteoporotic fractures. Polymorphisms in genes encoding PTH may contribute to genetic regulation of BMD and thus susceptibility to fracture risk. PTH stimulates the proliferation of osteoprogenitor cells, production of alkaline phosphatise, and bone matrix proteins that contribute to hard callus formation and increases strength at the site of fractured bone. During remodeling, PTH promotes osteoclastogenesis restoring the original shape, structure, and mechanical strength of the bone. Some PTH polymorphisms have shown an association with fracture risk. Further research is needed to elucidate the relative importance of PTH genetics and the mechanisms of genetic contributions to gene-gene interactions in the pathogenesis of osteoporosis and in fracture healing.

Dehydroepiandrosterone Stimulation of Osteoblastogenesis in Human MSCs Requires IGF-I Signaling.

Dehydroepiandrosterone (DHEA) is an adrenal steroid that circulates in high concentrations in humans in its sulfated form, DHEAS. Clinical and epidemiological studies suggested that low DHEAS levels may be associated with low bone mass. Previously, we and others showed that the effects of DHEA on the skeleton may be conferred partly by their ability to inhibit skeletal catabolic agents, for example, bone resorptive cytokine IL-6. In this study, we tested the hypothesis that the anabolic effects of DHEA on osteoblastogenesis require IGF-I signaling pathways. Using both primary cultures and a cell line of human bone marrow-derived mesenchymal stem cells (hMSCs), we show that DHEA and other steroids stimulate osteoblastogenesis as shown by alkaline phosphatase activity and osteoblast gene induction. The stimulation by DHEA on both IGF-I gene expression and osteoblastogenesis in hMSCs requires IGF-I receptor, PI3K, p38 MAPK, or p42/44 MAPK signaling pathways. This study adds information to indicate that DHEA may be useful for treating bone diseases through its inhibition of skeletal catabolic IL-6 and stimulation of anabolic IGF-I-mediated mechanisms. J. Cell. Biochem. 117: 1769-1774, 2016. © 2015 Wiley Periodicals, Inc.

Demineralized Bone and BMPs: Basic Science and Clinical Utility.

The clinical demand for bone void fillers led to the development of off-the-shelf banked bone and synthetic and biologic substitute materials to be used either alone or as bone graft volume extenders. Demineralized bone (DB) has a remarkable capacity to induce new bone formation even when implanted subcutaneously in experimental animals, a phenomenon termed "osteoinduction." DB products are now widely available through tissue bank procurement of bone from rigorously screened donors. When properly processed, DB products are useful in craniomaxillofacial, oral, hand, and orthopedic applications. The isolation of proteins believed to be responsible for the osteoinductive activity of DB, termed bone morphogenetic proteins (BMPs), led to the cloning of a family of genes and synthesis of recombinant human BMPs (rhBMPs). They have been approved for distribution and use in specific maxillofacial and orthopedic applications. Clinical trials and studies of orthopedic and craniofacial applications have indicated that supraphysiologic doses of a single recombinant protein are needed to promote bone repair. Information about the biology, chemistry, and actions of rhBMPs and DB has called into question whether a single recombinant BMP would result in clinically useful bone induction and morphogenesis. Compelling preclinical and specific clinical evidence has indicated the efficacy of DB and for rhBMPs either combined with autograft or compared with an autograft alone. In light of questions about potency and safety, however, additional high-level evidence is needed for specific clinical indications and appropriate patient populations that would benefit from their use.

Effect of age on regulation of human osteoclast differentiation.

Human skeletal aging is characterized as a gradual loss of bone mass due to an excess of bone resorption not balanced by new bone formation. Using human marrow cells, we tested the hypothesis that there is an age-dependent increase in osteoclastogenesis due to intrinsic changes in regulatory factors [macrophage-colony stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG)] and their receptors [c-fms and RANK]. In bone marrow cells (BMCs), c-fms (r = 0.61, P = 0.006) and RANK expression (r = 0.59, P = 0.008) were increased with age (27-82 years, n = 19). In vitro generation of osteoclasts was increased with age (r = 0.89, P = 0.007). In enriched marrow stromal cells (MSCs), constitutive expression of RANKL was increased with age (r = 0.41, P = 0.049) and expression of OPG was inversely correlated with age (r = -0.43, P = 0.039). Accordingly, there was an age-related increase in RANKL/OPG (r = 0.56, P = 0.005). These data indicate an age-related increase in human osteoclastogenesis that is associated with an intrinsic increase in expression of c-fms and RANK in osteoclast progenitors, and, in the supporting MSCs, an increase in pro-osteoclastogenic RANKL expression and a decrease in anti-osteoclastogenic OPG. These findings support the hypothesis that human marrow cells and their products can contribute to skeletal aging by increasing the generation of bone-resorbing osteoclasts. These findings help to explain underlying molecular mechanisms of progressive bone loss with advancing age in humans.

Effects of mouse genotype on bone wound healing and irradiation-induced delay of healing.

We tested the effects of mouse genotype (C57BL/6NHsd, NOD/SCID, SAMR1, and SAMP6) and ionizing irradiation on bone wound healing. Unicortical wounds were made in the proximal tibiae, and the time course of spontaneous healing and effects of irradiation were monitored radiographically and histologically. There was reproducible healing beginning with intramedullary osteogenesis, subsequent bone resorption by osteoclasts, gradual bridging of the cortical wound, and re-population of medullary hematopoietic cells. The most rapid wound closure was noted in SAMR1 mice, followed by SAMP6, C57BL/6NHsd, and NOD/SCID. Ionizing irradiation (20 Gy) to the leg significantly delayed bone wound healing in mice of all four genotypes. Mice with genetically-determined predisposition to early osteopenia (SAMP6) or with immune deficiency (NOD/SCID) had impairments in bone wound healing. These mouse models should be valuable for determining the effects of irradiation on bone healing and also for the design and testing of novel bone growth-enhancing drugs and mitigators of ionizing irradiation.

Gender differences in the growing, abnormal, and aging jaw.

Despite wide variations in the size and shape of the human face, head, and body, there is remarkable consistency for quantifiable gender-specific facial traits. The relationships between the growing jaws and tooth eruption are complex, but they show gender-specific trajectories in children and adolescents. Disturbances in genetic, endocrine, and nutritional regulatory controls result in gender-specific and nonspecific disorders. Gender-specific differences are also apparent in the aging jaw, with the acceleration of jawbone atrophy upon loss of teeth, especially in women.