W. G. Young

Tissue engineering for bone regeneration using differentiated alveolar bone cells in collagen scaffolds.

Regeneration of osseous defects by a tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. In this study the concept of tissue engineering was tested with collagen type I matrices seeded with cells with osteogenic potential and implanted into sites where osseous damage had occurred. Explant cultures of cells from human alveolar bone and gingiva were established. When seeded into a three-dimensional type I collagen-based scaffold, the bone-derived cells maintained their osteoblastic phenotype as monitored by mRNA and protein levels of the bone-related proteins including bone sialoprotein, osteocalcin, osteopontin, bone morphogenetic proteins 2 and 4, and alkaline phosphatase. These in vitro-developed matrices were implanted into critical-size bone defects in skulls of immunodeficient (SCID) mice. Wound healing was monitored for up to 4 weeks. When measured by microdensitometry the bone density within defects filled with osteoblast-derived matrix was significantly higher compared with defects filled with either collagen scaffold alone or collagen scaffold impregnated with gingival fibroblasts. New bone formation was found at all the sites treated with the osteoblast-derived matrix at 28 days, whereas no obvious new bone formation was identified at the same time point in the control groups. In situ hybridization for the human-specific Alu gene sequence indicated that the newly formed bone tissue resulted from both transplanted human osteoblasts and endogenous mesenchymal stem cells. The results indicate that cells derived from human alveolar bone can be incorporated into bioengineered scaffolds and synthesize a matrix, which on implantation can induce new bone formation.

Variability of the styloid process and stylohyoid ligament in panoramic radiographs

This article deals with the incidence of anatomic variations in the styloid process and stylohyoid ligament as found in panoramic radiographs. Also, the accuracy was determined for measuring the length of styloid processes from panoramic radiographs, the variation in length of the styloid process with age, and the relationship of elongated styloid processes and ossified stylohyoid ligaments to symptoms of styloid-stylohyoid syndrome. Variations in styloid process and stylohyoid ligaments are not uncommon, especially segmentation of the process in young subjects. The apparent length of the styloid process was magnified approximately 1.37 times and was never diminished by panoramic radiographs. A steady increase in styloid process length was observed to age 30 years, with another slight increase starting at age 60 years. Subjects with elongated styloid processes (greater than 40 mm) had the highest incidence of discomfort on swallowing, whereas subjects with ossification of the stylohyoid ligament had the highest incidence of discomfort on turning the head from side to side.

Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast–mineralized matrix complex. Histologically, at this time point a tissue structure with a “connective tissue”-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of immunodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.

Expression and Regulation of Insulin-Like Growth Factor-I in the Rat Incisor

Growth factors play an important role in the regulation of cell growth, division and differentiation. In this study the distribution and regulation of insulin-like growth factor-I (IGF-I) in the continuously erupting rat incisor was determined by immunohistochemistry. Results were evaluated both visually and with a computer-based image analysis system. The distribution and intensity of IGF-I immunoreactivity varied with developmental stage of the rat incisor. Strong IGF-I immunoreactivity was observed in differentiating odontoblasts and ameloblasts. The most intense immunoreactivity was observed in secretory ameloblasts, secretory odontoblasts and in maturation ameloblasts. Staining was weak or absent in post-secretory ameloblasts but persisted in post-secretory odontoblasts. Weak to moderate immunoreactivity was also seen in cells of the stratum intermedium and in the reduced enamel epithelium. Surrounding alveolar bone showed strong IGF-I immunoreactivity in osteoblasts and in the stratum basale and stratum spinosum of the adjacent labial gingival epithelium. In order to assess the role of GH in IGF-I expression, GH (65 micrograms/100 g bw) was administered for six days to dwarf GH deficient rats, producing a significant increase in body weight (P < 0.01). Measurements at different stages of odontogenesis showed that the staining intensity of secretory ameloblasts (P < 0.01) and maturation ameloblasts (P < 0.001) was significantly different between untreated and treated animals. These results indicate that IGF-I is present in cell populations of the enamel organ of the rat incisor found previously to exhibit growth hormone receptors, and that expression of IGF is GH dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

In Situ Hybridization Evidence for a Paracrine/Autocrine Role for Insulin-Like Growth Factor-I in Tooth Development

Insulin-like growth factor-I(IGF-I) has both metabolic and growth-promoting activities in many cell and tissue types. Although IGF-I is present in serum, it is also thought to have important autocrine and paracrine functions. Immunohistochemistry for IGF-I and its receptor have shown that IGF-I is synthesised locally by the tooth forming cells which exhibit both the IGF-I and the growth hormone receptors. This concept required to be tested by in situ hybridization. Using a digoxigenin-labelled synthetic oligodeoxyribonucleotide probe for IGF-I, we investigated the distribution of IGF-I mRNA in the continuously erupting rat incisor by in situ hybridization. The distribution and intensity of the hybridization signal varied with the developmental stage of the rat incisor. The cells of the apical loop expressed a positive hybridization signal, but the earliest polarised odontoblasts and pre-ameloblasts did not show any positive signal. The onset of enamel secretion was accompanied by a strong hybridization signal in the secretory ameloblasts as well as the odontoblasts. Maturation ameloblasts also demonstrated IGF-I message in their cytoplasm as well as their nuclei. The cells of the pulp and the dental follicle were consistently negative. However, in the adjacent alveolar bone, the signal was high in the osteoblasts and osteoclasts. These findings support the notion of paracrine or autocrine function for IGF-I in tooth development.

Growth hormone induces bone morphogenetic proteins and bone-related proteins in the developing rat periodontium.

The hypothesis that growth hormone (GH) up‐regulates the expression of enzymes, matrix proteins, and differentiation markers involved in mineralization of tooth and bone matrices was tested by the treatment of Lewis dwarf rats with GH over 5 days. The molar teeth and associated alveolar bone were processed for immunohistochemical demonstration of bone morphogenetic proteins 2 and 4 (BMP‐2 and ‐4), bone morphogenetic protein type IA receptor (BMPR‐IA), bone alkaline phosphatase (ALP), osteocalcin (OC), osteopontin (OPN), bone sialoprotein (BSP), and E11 protein (E11). The cementoblasts, osteoblasts, and periodontal ligament (PDL) cells responded to GH by expressing BMP‐2 and ‐4, BMPR‐IA, ALP, OC, and OPN and increasing the numbers of these cells. No changes were found in patterns of expression of the late differentiation markers BSP and E11 in response to GH. Thus, GH evokes expression of bone markers of early differentiation in cementoblasts, PDL cells, and osteoblasts of the periodontium. We propose that the induction of BMP‐2 and ‐4 and their receptor by GH compliments the role of GH‐induced insulin‐like growth factor 1 (IGF‐1) in promoting bone and tooth root formation.

Expression of growth hormone receptor by immunocytochemistry in rat molar root formation and alveolar bone remodeling

SummaryGrowth hormone (GH) may regulate tooth formation and bone remodeling associated with tooth eruption. This study reports the distribution of growth hormone receptor/binding protein in developing rat molars and adjacent alveolar bone by immunocytochemistry using well-characterized anti-growth hormone receptor monoclonal antibodies. These tissues represent an excellent model for studying the ontogenic changes that occur in odontogenic and osteogenic cells, as these cells are found in linear arrays displaying the various stages of morphological and functional differention, and differentiated function. Immunoreactivity was first seen in precementoblasts in contact with the epithelial root sheath, and preodontoblasts. However, growth hormone receptor immunoreactivity was associated primarily with the cytoplasm of odontogenic and osteogenic cells forming their respective matrices. Thus, cementoblasts and odontoblasts at sites of new matrix formation showed intense immunoreactivity whereas cementocytes and mature odontoblasts at later stages of tooth development were nonreactive. Osteoblasts engaged in intramembranous ossification in the alveolar bone were positive, although osteocytes and endosteal cells were immunonegative. Osteoclasts at sites of alveolar bone remodeling resorption were also immunopositive. These patterns of receptor expression parallel the ontogenic sequences of odontogenic and osteogenic cells and suggest that GH promotes the functional state of these cells. Our results also imply that GH may influence differentiation or differentiated functions associated with odontogenesis, osteogenesis, and bone remodeling independent of systemic insulin-like GF-I.

Comparison of the effects of growth hormone, insulin-like growth factor-I and fetal calf serum on mouse molar odontogenesis in vitro

The effects of growth hormone, its mediator insulin-like growth factor-I (IGF-I), and fetal calf serum on odontogenesis were compared to those of serum-free medium. Explanted, 16-day, fetal mouse first molar tooth germs in early bell stage were grown on semisolid, serum-free medium supplemented with ascorbic and retinoic acids. Recombinant human growth hormone at 50 or 100 ng/ml, IGF-I at 100 or 200 ng/ml, or fatal calf serum at 20% concentration were added to the media. Volumetric changes in serial sections of six tooth germs per treatment over 3 days of treatment (4, 5, 6 days in vitro) were compared by digitized morphometry. Mitotic indices were also compared and the cell densities of the dental papillae recorded. Qualitative ratings of differentiation were ascribed to each tooth germ by light microscopy. Differences in volume, mitotic activity and cell densities were found. The growth hormone-treated tooth germs were not larger than the serum-free ones but had increased mitotic indices and higher cell densities in the dental papillae. IGF-I-treated tooth germs had larger volumes than with all other treatments, e.g. germs treated with 200 ng/ml of IGF-I, after 6 days in culture, were significantly larger than with all other treatments (p<0.01-<0.001). Whilst IGF-I-treated germs displayed the greatest extent of differentiation, growth hormone-treated germs also showed advanced differentiation compared to those on serum-free medium. These results suggest that growth hormone and IGF-I are involved in odontogenesis of murine teeth in vitro by affecting mitotic activity, tissue volume and cell differentiation. In conjunction with previous immunohistochemical studies that show expression of growth hormone receptor and IGF-I in developing teeth, these results provide evidence that both growth hormones and its mediator play a part in odontogenesis.

Evidence for a local action of growth hormone in embryonic tooth development in the rat

Studies in non-dental embryonic tissues have suggested that an interaction between growth hormone and its receptor may play a role in growth and development before the foetal pituitary gland is competent. This study reports the distribution of growth hormone, its receptor and binding protein in developing rat tooth germs from embryonic day 17 to 21 and postnatal day 0 using antibodies specific for each of these proteins. Four foetal rats were processed at each time point (E17, E18, E20/21 and postnatal day 0). Following routine fixation and paraffin embedding, sections were treated with antisera to rat growth hormone, rat growth hormone binding protein and growth hormone receptor. Localization of antibody/antigen complexes was subsequently visualized by addition of biotinylated IgG and reaction with streptavidin peroxidase and diaminobenzidine. Assessment of the level of staining was qualitative and based on a subjective rankings ranging from equivocal to very strong staining. Overall, growth hormone and its binding protein were located both in the cellular elements and throughout the extracellular matrix, whereas the growth hormone receptor showed an exclusively intra-cellular location. All three proteins were detectable in cells of the dental epithelium and mesenchyme at the primordial bud stage (E17) which occurs prior to expression of pituitary growth hormone. At the cap stage of odontogenesis (E18-19), numerous cells in both the dental epithelium and mesenchyme were intensely immunoreactive for growth hormone, its binding protein and receptor. In the succeeding early bell stage (E20-21), most of the mesenchymal cells in the dental pulp were mildly positive for these proteins, while the dental epithelium and adjacent mesenchyme were more immunoreactive. At the late bell stage (postnatal day 0), all three proteins were localized in dental epithelium, differentiating mesenchymal cells the cuspal surface facing the epithelial-mesenchymal interface, preodontoblasts, and odontoblasts forming dentine. From these observations, immunoreactive growth hormone, its receptor and binding protein appear to be expressed in odontogenic cells undergoing histodifferentiation, morphodifferentiation and dentinogenesis in a cell-type and stage-specific pattern throughout embryonic tooth development. This suggests the possibility that growth hormone, or a growth hormone-like protein, plays a paracrine/autocrine role in tooth development in utero.

Immunocytochemical localization of growth hormone receptor in rat maxillary teeth.

To address the question of what role growth hormone may have in stimulating tooth formation, the distribution of its receptor/binding protein in developing rat incisors and molars was studied immunocytochemically using well-characterized monoclonal antibodies. Ten female 45-day-old Wistar rats were perfused with 4% paraformaldehyde. Five-microns paraffin sections of the growing end of maxillary incisors and molars were cut, deparaffinized and incubated with mouse anti-growth hormone receptor antibodies or control antibodies. A three-layer streptavidin peroxidase technique was used to detect bound antibody. Immunoreaction product was associated primarily with the cytoplasm of cells at certain stages of differentiation. Dividing cells, differentiating preameloblasts and preodontoblasts, secretory ameloblasts and odontoblasts showed immunoreactivity. Undifferentiated dental epithelium cells, stellate reticulum, external dental epithelial cells, mature odontoblasts, and most of cells in the dental papilla were non-reactive. However, at certain stages of tooth development, the stratum intermedium and the external dental epithelium also stained positively. The presence of growth hormone receptor/binding protein in tooth cells at different stages of their development indicates that growth hormone may influence cell proliferation, differentiation and differentiated functions of ameloblasts, odontoblasts and cementoblasts independent of a systemic mediator, and thus may be involved in stimulating odontogenesis directly.