P. Gehron Robey

Stem Cell Properties of Human Dental Pulp Stem Cells

In this study, we characterized the self-renewal capability, multi-lineage differentiation capacity, and clonogenic efficiency of human dental pulp stem cells (DPSCs). DPSCs were capable of forming ectopic dentin and associated pulp tissue in vivo. Stromal-like cells were reestablished in culture from primary DPSC transplants and re-transplanted into immunocompromised mice to generate a dentin-pulp-like tissue, demonstrating their self-renewal capability. DPSCs were also found to be capable of differentiating into adipocytes and neural-like cells. The odontogenic potential of 12 individual single-colony-derived DPSC strains was determined. Two-thirds of the single-colony-derived DPSC strains generated abundant ectopic dentin in vivo, while only a limited amount of dentin was detected in the remaining one-third. These results indicate that single-colony-derived DPSC strains differ from each other with respect to their rate of odontogenesis. Taken together, these results demonstrate that DPSCs possess stem-cell-like qualities, including self-renewal capability and multi-lineage differentiation.

Enzyme-linked immunoassay (ELISA) for connective tissue components

Abstract Enzyme-linked immunoadsorbant assays have been developed for types I, II, III, and IV collagen and for laminin and fibronectin. These assays offer a specific, sensitive, and convenient method for the measurement of various connective tissue components either separately or simultaneously. Plastic microtiter wells are coated with purified antigen, then antibodies to the antigen are allowed to bind to the insolubilized antigen in each well. The amount of bound antibody is determined by incubation with a second antibody which is covalently linked to alkaline phosphatase or horseradish peroxidase. The amount of bound enzyme is assayed after adding an appropriate substrate. The level of protein in a sample is estimated from its ability to inhibit the binding of the first antibody to the antigen-coated well. Specificity of the assay depends on the purity of the adsorbed antigen and allows for highly specific tests. Under optimum conditions the sensitivity of the assay is determined by the KB of the antibody and allows 10–20 ng of a specific type of collagen or laminin and 1 ng fibronectin to be detected.

Comparison of Stem-cell-mediated Osteogenesis and Dentinogenesis

The difference between stem-cell-mediated bone and dentin regeneration is not yet well-understood. Here we use an in vivo stem cell transplantation system to investigate differential regulation mechanisms of bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). Elevated expression of basic fibroblast growth factor (bFGF) and matrix metalloproteinase 9 (MMP-9, gelatinase B) was found to be associated with the formation of hematopoietic marrow in BMSSC transplants, but not in the connective tissue of DPSC transplants. The expression of dentin sialoprotein (DSP) specifically marked dentin synthesis in DPSC transplants. Moreover, DPSCs were found to be able to generate reparative dentin-like tissue on the surface of human dentin in vivo. This study provided direct evidence to suggest that osteogenesis and dentinogenesis mediated by BMSSCs and DPSCs, respectively, may be regulated by distinct mechanisms, leading to the different organization of the mineralized and non-mineralized tissues.

Phenotypic Effects of Biglycan Deficiency Are Linked to Collagen Fibril Abnormalities, Are Synergized by Decorin Deficiency, and Mimic Ehlers-Danlos-Like Changes in Bone and Other Connective Tissues†

Decorin (dcn) and biglycan (bgn), two members of the family of small leucine‐rich proteoglycans (SLRPs), are the predominant proteoglycans expressed in skin and bone, respectively. Targeted disruption of the dcn gene results in skin laxity and fragility, whereas disruption of the bgn gene results in reduced skeletal growth and bone mass leading to generalized osteopenia, particularly in older animals. Here, we report that bgn deficiency leads to structural abnormality in collagen fibrils in bone, dermis, and tendon, and to a “subclinical” cutaneous phenotype with thinning of the dermis but without overt skin fragility. A comparative ultrastructural study of different tissues from bgn‐ and dcn‐deficient mice revealed that bgn and dcn deficiency have similar effects on collagen fibril structure in the dermis but not in bone. Ultrastructural and phenotypic analysis of newly generated bgn/dcn double‐knockout (KO) mice revealed that the effects of dcn and bgn deficiency are additive in the dermis and synergistic in bone. Severe skin fragility and marked osteopenia characterize the phenotype of double‐KO animals in which progeroid changes are observed also in the skin. Ultrastructural analysis of bone collagen fibrils in bone of double‐KO mice reveals a complete loss of the basic fibril geometry with the emergence of marked “serrated fibril” morphology. The phenotype of the double‐KO animal mimics directly the rare progeroid variant of human Ehlers‐Danlos syndrome (EDS), in which skin fragility, progeroid changes in the skin (reduced hypodermis), and osteopenia concur as a result of impaired glycosaminoglycan (GAG) linking to bgn and dcn core proteins. Our data show that changes in collagen fibril morphology reminiscent of those occurring in the varied spectrum of human EDS are induced by both bgn deficiency and dcn deficiency in mice. The effects of an individual SLRP deficiency are tissue specific, and the expression of a gross phenotype depends on multiple variables including level of expression of individual SLRPs in different tissues and synergisms between different SLRPs (and likely other macromolecules) in determining matrix structure and functional properties.

Bone Marrow Stromal Cells: Characterization and Clinical Application:

The bone marrow stroma consists of a heterogeneous population of cells that provide the structural and physiological support for hematopoietic cells. Additionally, the bone marrow stroma contains cells with a stem-cell-like character that allows them to differentiate into bone, cartilage, adipocytes, and hematopoietic supporting tissues. Several experimental approaches have been used to characterize the development and functional nature of these cells in vivo and their differentiating potential in vitro. In vivo, presumptive osteogenic precursors have been identified by morphologic and immunohistochemical methods. In culture, the stromal cells can be separated from hematopoietic cells by their differential adhesion to tissue culture plastic and their prolonged proliferative potential. In cultures generated from single-cell suspensions of marrow, bone marrow stromal cells grow in colonies, each derived from a single precursor cell termed the colony-forming unit-fibroblast. Culture methods have been developed to expand marrow stromal cells derived from human, mouse, and other species. Under appropriate conditions, these cells are capable of forming new bone after in vivo transplantation. Various methods of cultivation and transplantation conditions have been studied and found to have substantial influence on the transplantation outcome. The finding that bone marrow stromal cells can be manipulated in vitro and subsequently form bone in vivo provides a powerful new model system for studying the basic biology of bone and for generating models for therapeutic strategies aimed at regenerating skeletal elements.

Changes in apatite crystal size in bones of patients with osteogenesis imperfecta

SummaryApatite crystal size in compact bone of children (age<11 years) and adolescents (age>12 years) with osteogenesis imperfecta (OI) was analyzed by X-ray diffraction. Eight type I, 4 type II, 11 type III, and 14 type IV OI patients were studied along with 9 controls. The crystal size was most significantly reduced in type II patients, all of whom had died at birth. Crystal size was also diminished in both children and adolescents with types III and IV, whereas with type I OI, crystal size was reduced in children only, returning to normal in adolescence. There was a trend toward increased bone crystal size with age in both OI patients and controls.

Species Differences in Growth Requirements for Bone Marrow Stromal Fibroblast Colony Formation In Vitro

Abstract. The marrow stromal fibroblast (MSF) population has been shown to include precursor cells for at least five types of connective tissue: bone, cartilage, adipose tissue, fibrous tissue, and hematopoiesis-supporting reticular stroma. In this study, growth requirements for MSF colony formation were studied in vitro. In order to exclude the influence of nonadherent cells, after a period of initial adhesion of bone marrow cells in serum-containing medium nonadherent cells were removed. Further cultivation was carried out in either serum-containing or serum-free conditions, with or without feeder cells (irradiated bone marrow cells). This approach revealed differences between animal species in initial MSF growth requirements. In serum-containing conditions, mouse MSF precursor cells (colony-forming units-fibroblast, CFU-Fs) were shown to be feeder cell dependent: MSF colonies were formed only in the presence of feeder cells. Guinea pig CFU-Fs were partially feeder cell dependent, whereas human CFU-Fs were feeder cell independent. In serum-free conditions, CFU-Fs of all three species were feeder cell dependent. The difference between the growth requirements for mouse and human MSFs was not caused by serum origin or concentration, feeder cell origin, or differences in the preparation of marrow cell suspensions.

Osteoclastogenesis in fibrous dysplasia of bone: in situ and in vitro analysis of IL-6 expression

Fibrous dysplasia of bone (FD) is caused by somatic mutations of the GNAS1 gene, which lead to constitutive activation of adenylyl cyclase and overproduction of cAMP in osteogenic cells. Previous in vitro studies using nonclonal, heterogeneous strains of FD-derived cells suggested that IL-6 might play a critical role in promoting excess osteoclastogenesis in FD. In this study, we investigated IL-6 expression in FD in situ and its relationship to the actual patterns of osteoclastogenesis within the abnormal tissue. We found that osteoclastogenesis is not spatially restricted to bone surfaces in FD but occurs to a large extent ectopicly in the fibrous tissue, where stromal cells diffusely express IL-6 mRNA and exhibit a characteristic cell morphology. We also observed specific expression of IL-6 mRNA in a proportion of osteoclasts, suggesting that an autocrine/paracrine loop may contribute to osteoclastogenesis in vivo in FD, as in some other bone diseases, including Pagets disease. We also generated homogeneous, clonally derived strains of wild-type and GNAS1-mutated stromal cells from the same individual, parent FD lesions. In this way, we could show that mutated stromal cells produce IL-6 at a basal magnitude and rate that are significantly higher than in the cognate wild-type cells. Conversely, wild-type cells respond to db-cAMP with a severalfold increase in magnitude and rate of IL-6 production, whereas mutant strains remain essentially unresponsive. Our data establish a direct link between GNAS1 mutations in stromal cells and IL-6 production but also define the complexity of the role of IL-6 in regulating osteoclastogenesis in FD in vivo. Here, patterns of osteoclastogenesis and bone resorption reflect not only the cell-autonomous effects of GNAS1 mutations in osteogenic cells (including IL-6 production) but also the local and systemic context to which non-osteogenic cells, local proportions of wild-type vs mutated cells, and systemic hormones contribute.

The anatomy of bone sialoprotein immunoreactive sites in bone as revealed by combined ultrastructural histochemistry and immunohistochemistry.

Bone sialoprotein was immunolocalized at the EM level in thin Lowicryl K4M sections of rat bone. Because of the unconventional EM morphology of the bone matrix seen in thin demineralized acrylate sections, the pattern of immunolabeling was compared with detailed structural images of demineralized bone obtained using an en bloc treatment of tissue samples with the cationic electron ‘dye’, Malachite Green (MG), which provides stabilization and retention of anionic material throughout specimen processing. A system of structures corresponding to the sites of bone sialoprotein (BSP) immunoreactivity, as seen in Lowicryl K4M thin sections, could be readily identified in the MG-treated, expoxy thin sections. This system includes the cement lines, and aggregates of similar material within mineralized bone and mineralizing osteoid. The virtual identity of BSP distribution with the arrangement of the MG-visualized material indicates that a BSP-enriched, noncollagenous phase can be demonstrated using different, unrelated tissue preparation and imaging protocols for EM. Besides improving our understanding of the distribution of bone sialoprotein in bone, these data assign a previously unrecognized structural dimension to noncollagenous material in the bone matrix.

Production and characterization of an antibody against the human bone GLA protein (BGP/osteocalcin) propeptide and its use in immunocytochemistry of bone cells.

We have generated and characterized an antibody that recognizes the C-terminal sequence of the propeptide of human bone GLA protein (BGP/osteocalcin)(amino acid -26 to -1, with +1 being the amino terminus of the mature protein). The range of sensitivity of the antibody, as determined by enzyme-linked immunosorbent assay (ELISA), was 0.5-250 ng/ml. The antibody effectively recognized pro-BGP in cell layer extracts of transformed cells (KT-005), but did not recognize mature, propeptide-less BGP in the medium from the same cultures. Strong labelling was obtained using this antibody in immunoperoxidase staining or immunofluorescence of both transformed and normal human bone cells in vitro. Monensin significantly altered the intracellular pattern of labelling in immunofluorescence studies, indicating that the recognized antigen was associated with the cellular secretory pathway. We also obtained a specific and strong staining of cells in tissue sections of human fetal bone. Antibodies against the mature protein strongly stained the mineralization front, but did not stain cells to any appreciable level. Newly embedded osteocytes were the predominant cell type stained in such material, suggesting that they may represent the major of BGP in the intact tissue. These observations indicate that BGP synthesis is a late event in osteoblastic development and that antibodies generated against the propeptide sequence are a potentially powerful tool in the analysis of bone tumors and evaluation of osteoblastic differentiation.