Biomedical subjects

Kurt D Hankenson

Publications and source records attributed to Kurt D Hankenson.

16 recordsLinked to original sources

PubMed2006

Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6.

Bone marrow-derived mesenchymal stem cells (MSC) are multipotent, self-renewing, mesodermal-origin stem cells that are sequestered in the endosteal compartment. MSC are maintained in a relative state of quiescence in vivo but in response to a variety of physiological and pathological stimuli, proliferate and differentiate into osteoblasts, chondrocytes, adipocytes, or hematopoiesis-supporting stromal cells. Little is understood regarding the cellular or molecular events underlying MSC fate decisions. We report that human MSC (hMSC) cultured in defined, serum-free conditions respond to a narrow spectrum of growth factors with osteogenic commitment, differentiation, and hydroxyapatite deposition. Of the osteogenic factors we examined, only treatment with bone morphogenetic protein (BMP) results in osteoinduction under defined serum-free conditions. Among BMP-2, 4, 6, and 7, BMP-6 is the most consistent and potent regulator of osteoblast differentiation and, of these BMPs, only BMP-6 gene expression is detected prior to hMSC osteoblast differentiation. Addition of exogenous BMP-6 to hMSC induces the expression or upregulation of a repertoire of osteoblast-related genes including type I collagen, osteocalcin, bone sialoprotein, and their regulatory transcription factors Cbfa1/Runx2, and Osterix. This translates into increased production of osteogenic extracellular matrix (ECM) with subsequent hydroxyapatite deposition.

Alkaline Phosphatase↗

PubMed2006

Reduced expression of thrombospondins and craniofacial dysmorphism in mice overexpressing Fra1.

UNLABELLED: Fra1 transgenic (Tg) mice develop osteosclerosis and exhibit altered expression of bone matrix proteins. We found that expression of Thbs1 and Thbs2 was reduced in Fra1 Tg osteoblasts. Fra1 Tg and non-osteosclerotic Thbs1-/-Thbs2-/- mice share an edge-to-edge bite. Therefore, reduced expression of thrombospondins may contribute to craniofacial dysmorphism independently of osteosclerosis. INTRODUCTION: Tg mice overexpressing Fra1, a component of the transcription factor activator protein-1 (AP-1), show progressive osteosclerosis caused by cell autonomous abnormalities in osteoblasts. The expression of several bone matrix proteins, including matrix gla protein, is dysregulated in Fra1 Tg osteoblasts. MATERIALS AND METHODS: In osteoblastogenic cultures, altered bone matrix production by Fra1 overexpression was monitored using Alizarin red staining, quantitative RT-PCR, and Western blotting. Responsiveness to ovariectomy was examined by bone histomorphometry. Craniofacial parameters were measured on radiographs and using CT. RESULTS: Thrombospondin-1 (Thbs1) and thrombospondin-2 (Thbs2) were reduced in Fra1 Tg osteoblasts differentiated in vitro and in bones from Fra1 Tg mice. Despite alterations in bone matrix proteins, ovariectomy induces high turnover bone loss in Fra1 Tg mice as in wildtype mice. Fra1 Tg mice, as well as Thbs1-/- Thbs2-/- mice, which do not show osteosclerosis, exhibit an edge-to-edge bite phenotype associated with craniofacial dysmorphism. CONCLUSIONS: These data suggest that reduced expression of thrombospondins in Fra1 Tg mice underlies craniofacial dysmorphism, independent of osteosclerosis.

Animals↗

PubMed2006

Matricellular proteins: Extracellular modulators of bone development, remodeling, and regeneration.

Matricellular proteins are components of the extracellular matrix which are highly expressed in the developing and mature skeleton. Members of this protein class serve as biological mediators of cell function by interacting directly with cells or by modulating the activity of growth factors, proteases, and other extracellular matrix proteins. Although skeletons of matricellular protein-null mice are grossly normal, they each display unique deficiencies that are often magnified under pathological conditions. In addition, bone cells from wild-type and matricellular protein-null mice behave differently in various in vitro models of bone matrix synthesis and turnover. In this review, osteopontin, bone sialoprotein, tenascin C, SPARC, and thrombospondins 1 and 2 will each be discussed in the context of bone cell biology. Because the biological effects of matricellular proteins are largely context dependent, in vivo and in vitro results must be considered together in order to fully appreciate the specific contributions that matricellular proteins make to bone physiology and pathophysiology. In particular, it is clear that although matricellular proteins are not required for bone development and function, the proteins act to modulate post-natal bone structure in response to aging, ovariectomy, mechanical loading, and bone regeneration.

Animals↗

PubMed2006

Novel explant model to study mechanotransduction and cell-cell communication.

To understand in situ behavior of osteocytes, we characterized a model of osteocytes in their native bone matrix and demonstrated real-time biologic activity of osteocytes while bending the bone matrix. Using 43 male Sprague-Dawley rats, dumbbell-shaped explants were harvested from stainless steel femoral implants after 6-12 weeks and incubated in culture medium or fixed. Sixteen specimens were used to determine bone volume density (BV/TV), volumetric bone mineral density (BMD) and histology for different implantation periods. Osteocyte viability was evaluated by L-lactate dehydrogenase (LDH) activity in 12 cultured explants. Confocal microscopy was used to assess tracer diffusion in three explants and changes in osteocyte pH of a mechanically loaded explant. From 6 to 12 weeks, explant BV/TV and volumetric BMD trended up 92.5% and 101%, respectively. They were significantly and highly correlated. Tissues were uniformly intramembranous and all bone cell types were present. Explants maintained LDH activity through culture day 8. Diffusion at 200 microM was limited to 1,209 Da. Explants appeared capable of reproducing complex bone biology. This model may be useful in understanding osteocyte mechanotransduction in the context of a physiologically relevant bone matrix.

Animals↗

PubMed2006

Pleiotropic phenotype of a genomic knock-in of an RGS-insensitive G184S Gnai2 allele.

Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the Galpha(i2) subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The Galpha(i2)(G184S) knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous Galpha(i2)(+/G184S) mice show some but not all of these abnormalities. Thus, loss of RGS actions at Galpha(i2) produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts.

Alleles↗

PubMed2005

Mice lacking thrombospondin 2 show an atypical pattern of endocortical and periosteal bone formation in response to mechanical loading.

Thrombospondin 2 (TSP2) is an extracellular matrix (ECM) protein localized to bone. Since mice with a targeted disruption of the TSP2 gene (TSP2-null) have increased bone formation, we hypothesized that mice lacking TSP2 would show an enhanced osteogenic response to mechanical loading. We addressed our hypothesis by subjecting wild-type (WT) and TSP2-null mice to mechanical loading using the non-invasive murine tibia loading device, and statistical comparisons were made between loaded and unloaded bones within genotype, between genotypes, and between the periosteal and endocortical surfaces within genotype. Right tibiae of WT and TSP2-null mice received 5 days of a low-magnitude loading protocol. This low-magnitude loading (inducing approximately 900 and 500 muepsilon at periosteal and endocortical surfaces of WT bones, respectively) affected neither periosteal nor endocortical bone formation rate (BFR/BS) when comparing loaded to intact bones in either WT or TSP2-null mice, nor did it result in any significant differences between WT and TSP2-null. As well, there was no difference between loaded endocortical and periosteal surfaces in WT mice; however, endocortical BFR/BS in TSP2-null loaded tibia was significantly elevated relative to the periosteal BFR/BS-despite peak periosteal strains being significantly greater than endocortical strains in TSP2-null mice (690 versus 460 muepsilon). To confirm this counterintuitive surface-specific response in TSP2-null mice and to induce significant periosteal bone formation, osteogenic potency of the loading protocol was amplified by doubling the number of loading bouts (10 loading days) and loading magnitude (1 Hz, resulting in 1400 and 900 muepsilon peak strain at the periosteal and endocortical surfaces, respectively). Under load, both WT and TSP2-null mice showed significantly increased periosteal mineralizing surface (by nearly three-fold and five-fold, respectively), but mineral apposition rate (MAR) was not statistically changed. The increased MS/BS resulted in a five-fold increase in WT periosteal BFR/BS, but the TSP2-null periosteal BFR/BS was unchanged. Furthermore, this increase in WT loaded periosteal BFR/BS was statistically greater than the WT endocortical BFR/BS. At the endocortical surface of WT mice, loading did not significantly increase bone formation parameters (versus intact). In contrast, at the endocortical surface of TSP2-null mice, loading induced a significant two-fold increase in BFR/BS (versus intact), that was also significantly greater than the endocortical BFR/BS of loaded WT mice. Thus, exogenous loading of TSP2-null mice resulted in highly variable responses that did not reflect the induced strains at the periosteal and endocortical surfaces. While in WT mice, loading resulted in increased periosteal BFR/BS that was greater than the endocortical BFR/BS, in TSP2-null mice loading resulted in endocortical (not periosteal) BFR/BS that was elevated. This reversal in envelope-specific bone formation in TSP2-null mice occurred despite periosteal strains being significantly greater than endocortical (1290 versus 775 muepsilon) and strain distributions being similar to that of WT. These results show that the disruption of a single gene can lead to a reversal in normal pattern of load induced bone formation, and more specifically, that the functional interaction of TSP2 with mechanical loading is highly contextual and specific to the cortical bone envelope examined.

Animals↗

PubMed2005

Regulation of osteoblastogenesis and bone mass by Wnt10b.

Wnts comprise a family of secreted signaling proteins that regulate diverse developmental processes. Activation of Wnt signaling by Wnt10b inhibits differentiation of preadipocytes and blocks adipose tissue development; however, the effect of Wnt10b on other mesenchymal lineages has not been defined. To explore the physiological role of Wnt signaling in bone development, we analyzed FABP4-Wnt10b mice, which express the Wnt10b transgene in marrow. Femurs from FABP4-Wnt10b mice have almost four times as much bone in the distal metaphyses and are mechanically stronger. These mice maintain elevated bone mass at least through 23 months of age. In addition, FABP4-Wnt10b mice are protected from the bone loss characteristic of estrogen deficiency. We used pharmacological and genetic approaches to demonstrate that canonical Wnt signaling stimulates osteoblastogenesis and inhibits adipogenesis of bipotential mesenchymal precursors. Wnt10b shifts cell fate toward the osteoblast lineage by induction of the osteoblastogenic transcription factors Runx2, Dlx5, and osterix and suppression of the adipogenic transcription factors C/EBPalpha and PPARgamma. One mechanism whereby Wnt10b promotes osteoblastogenesis is suppression of PPARgamma expression. Finally, Wnt10b-/- mice have decreased trabecular bone and serum osteocalcin, confirming that Wnt10b is an endogenous regulator of bone formation.

Aging↗

PubMed2005

A simple method for stem cell labeling with fluorine 18.

Hexadecyl-4-[(18)F]fluorobenzoate ([(18)F]HFB), a long chain fluorinated benzoic acid ester, was prepared in a one-step synthesis by aromatic nucleophilic substitution of [(18)F]fluoride ion on hexadecyl-4-(N,N,N-trimethylammonio)benzoate. The radiolabeled ester was obtained in good yields (52% decay corrected) and high purity (97%). [(18)F]HFB was used to radiolabel rat mesenchymal stem cells (MSCs) by absorption into cell membranes. MicroPET imaging of [(18)F]HFB-labeled MSCs following intravenous injection into the rat showed the expected high and persistent accumulation of radioactivity in the lungs. [(18)F]HFB is thus simple to prepare and uses labeling agent for short-term distribution studies of injected stem cells.

Animals↗

PubMed2005

Mice with a disruption of the thrombospondin 3 gene differ in geometric and biomechanical properties of bone and have accelerated development of the femoral head.

Thrombospondin 3 (TSP3) is structurally similar to cartilage oligomeric matrix protein (COMP/TSP5), but its function is unknown. To determine the functional significance of TSP3, we generated mice with a targeted disruption of Thbs3. TSP3-null mice are viable and fertile and show normal prenatal skeletal patterning, based on Alcian blue/Alizarin red S staining. However, subtle and transient abnormalities were detected in the developing postnatal skeleton. Young adult TSP3-null mice are heavier than controls, and analyses of the geometric and biomechanical properties of long bones show increases in the moments of inertia, endocortical and periostal radii, and failure load. The bones of 9-week-old TSP3-null male mice also have a significantly greater cortical area. Most of these differences were no longer detected in 15-week-old mice. Micro-computed tomography scans showed that the trabecular bone proximal to the femoral head growth plate developed at an earlier time in TSP3-null mice than in wild-type mice. Thus, vascular invasion and ossification start in the femoral heads of TSP3-null mice at 9 weeks, whereas the wild-type femoral head is still composed of hypertrophic chondroctyes in a calcified matrix at 15 weeks. These results provide evidence for a role for TSP3 in the regulation of skeletal maturation in mice.

Animals↗

PubMed2004

Huntingtin Interacting Protein 1 mutations lead to abnormal hematopoiesis, spinal defects and cataracts.

Huntingtin Interacting Protein 1 (HIP1) binds clathrin and AP2, is overexpressed in multiple human tumors, and transforms fibroblasts. The function of HIP1 is unknown although it is thought to play a fundamental role in clathrin trafficking. Gene-targeted Hip1-/- mice develop premature testicular degeneration and severe spinal deformities. Yet, although HIP1 is expressed in many tissues including the spleen and bone marrow and was part of a leukemogenic translocation, its role in hematopoiesis has not been examined. In this study we report that three different mutations of murine Hip1 lead to hematopoietic abnormalities reflected by diminished early progenitor frequencies and resistance to 5-FU-induced bone marrow toxicity. Two of the Hip1 mutant lines also display the previously described spinal defects. These observations indicate that, in addition to being required for the survival/proliferation of cancer cells and germline progenitors, HIP1 is also required for the survival/proliferation of diverse types of somatic cells, including hematopoietic progenitors.

Animals↗

PubMed2004

Gene targeting in stem cells from individuals with osteogenesis imperfecta.

Adult stem cells offer the potential to treat many diseases through a combination of ex vivo genetic manipulation and autologous transplantation. Mesenchymal stem cells (MSCs, also referred to as marrow stromal cells) are adult stem cells that can be isolated as proliferating, adherent cells from bones. MSCs can differentiate into multiple cell types present in several tissues, including bone, fat, cartilage, and muscle, making them ideal candidates for a variety of cell-based therapies. Here, we have used adeno-associated virus vectors to disrupt dominant-negative mutant COL1A1 collagen genes in MSCs from individuals with the brittle bone disorder osteogenesis imperfecta, demonstrating successful gene targeting in adult human stem cells.

Alleles↗

PubMed2004

Animal models of tendon and ligament injuries for tissue engineering applications.

Animal modeling continues to be an important component to the critical evaluation of new techniques to treat tendon and ligament injuries. Appropriate choice and analysis of these models is essential. Factors that should be considered in assessing a potential model include the tissue type (ligament versus tendon and intrasynovial versus extrasynovial), the type of injury to be modeled, the techniques of measuring the response to treatment, the ease of comparison to previously published models and finally, the ease of translation to human trials and eventually to clinical usage.

Animals↗

PubMed2003

Megakaryocytes require thrombospondin-2 for normal platelet formation and function.

Mice that lack the matricellular angiogenesis inhibitor, thrombospondin-2 (TSP2), display a bleeding diathesis, despite normal blood coagulation and the lack of thrombocytopenia. Although platelets do not contain detectable levels of TSP2, TSP2-null platelets are compromised in their ability to aggregate in vivo in response to denudation of the carotid artery endothelium, and in vitro following exposure to adenosine diphosphate (ADP). Megakaryocytes (MKs) show high levels of TSP2 by immunohistochemical analysis of bone marrow. However, when cultured in vitro, MKs contain little TSP2 protein or mRNA. These findings suggest that most TSP2 is acquired from the bone marrow microenvironment. Consistent with this hypothesis, MKs take up recombinant TSP2 in an integrin-dependent manner when it is supplied in the culture medium. Furthermore, uptake of TSP2 in vitro affects MK differentiation and proplatelet formation. The functional significance of this process is supported by the presence of ultrastructural abnormalities in TSP2-null bone marrow, including extensive fragmentation of the peripheral zone in MKs and failure of this zone to form close associations with vascular sinuses. We conclude that the uptake of TSP2 by MKs from the marrow milieu is required for proper MK function and the release of functionally competent platelets.

Animals↗

PubMed2002

Diminished callus size and cartilage synthesis in alpha 1 beta 1 integrin-deficient mice during bone fracture healing.

Integrins mediate cell adhesion to extracellular matrix components. Integrin alpha 1 beta 1 is a collagen receptor expressed on many mesenchymal cells, but mice deficient in alpha 1 integrin (alpha1-KO) have no gross structural defects. Here, the regeneration of a fractured long bone was studied in alpha1-KO mice. These mice developed significantly less callus tissue than the wild-type (WT) mice, and safranin staining revealed a defect in cartilage formation. The mRNA levels of nine extracellular matrix genes in calluses were evaluated by Northern blotting. During the first 9 days the mRNA levels of cartilage-related genes, including type II collagen, type IX collagen, and type X collagen, were lower in alpha1-KO mice than in WT mice, consistent with the reduced synthesis of cartilaginous matrix appreciated in tissue sections. Histological observations also suggested a diminished number of chondrocytes in the alpha 1-KO callus. Proliferating cell nuclear antigen staining revealed a reduction of mesenchymal progenitors at the callus site. Although, the number of mesenchymal stem cells (MSCs) obtained from WT and alpha 1-KO whole marrow was equal, in cell culture the proliferation rate of the MSCs of alpha 1-KO mice was slower, recapitulating the in vivo observation of reduced callus cell proliferation. The results demonstrate the importance of proper collagen-integrin interaction in fracture healing and suggest that alpha1 integrin plays an essential role in the regulation of MSC proliferation and cartilage production.

Aggrecans↗

PubMed2002

Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism.

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.

Animals↗

PubMed2002

The secreted protein thrombospondin 2 is an autocrine inhibitor of marrow stromal cell proliferation.

Marrow stromal cells (MSCs) are obtained in increased number from mice in which the thrombospondin 2 (TSP2) gene is disrupted, and these cells show increased DNA synthesis in vitro. To examine more closely the role of TSP2 in the physiology and osteogenic differentiation of MSCs, an in-depth characterization of TSP2-null MSCs was conducted. Determination of TSP2 protein content by Western analysis and RNA levels by reverse-transcription polymerase chain reaction (RT-PCR) indicated that MSCs are the primary source of TSP2 in the marrow and secrete abundant TSP2 into culture medium. Morphologically, the TSP2-null and wild-type (WT) cell populations were similar and by flow cytometry contained equivalent numbers of CD44+, Mac1+, intercellular adhesion molecule-1 (ICAM-1+), and ScaI+ cells. TSP2-null cells showed delayed mineralization associated with an increased rate of proliferation. Consistent with this finding, there was a decrease in expression of collagen and osteocalcin RNA by TSP2-null MSCs on day 7 and increased osteopontin expression on day 7 and day 14. In add-back experiments, recombinant TSP2 produced a dose-dependent decrease in proliferation. This reduction was associated with an accumulation of TSP2-treated cells in the G1 phase of the cell cycle and did not result from an increase in apoptosis. When TSP2 treatment was terminated, the cell population reentered the S phase. We conclude that the increased endosteal bone formation observed in TSP2-null mice results primarily from the failure of TSP2 to regulate locally MSC cell cycle progression.

Animals↗