Transplantation of labeled periodontal ligament cells promotes regeneration of alveolar bone.

Regeneration of damaged periodontal tissues is mediated by periodontal cells, but a major sub-population comprises highly differentiated cells that do not renew. To overcome the loss of specialized cell types caused by disease, various therapeutic approaches including cell transplants have been developed to promote cell re-population in periodontal tissues. As previous transplantation studies used unlabeled cells, that are indistinguishable from host cells, it has been difficult to assess the contributions of transplanted cells to the healing processes. To track the fate and differentiation of rat periodontal cells transplanted into periodontal wounds, we used collagen-coated fluorescent beads as a permanent endocytosed marker, or cells constitutively expressing beta-galactosidase. We assessed osteogenic cell differentiation with immunohistochemical staining for osteopontin and bone sialoprotein. Cells were transplanted into periodontal wounds created in Sprague--Dawley male rats that are null for beta-galactosidase. Defects were allowed to heal spontaneously (controls), or were closed with collagen implants mixed with beta-galactosidase-positive (Lac-Z) periodontal cells, or closed with collagen implants mixed with periodontal cells loaded with fluorescent beads. Animals were killed at 1 and 2 weeks after surgery and tissues were prepared for morphometric assessment and immunostaining for osteopontin (OPN) and bone sialoprotein (BSP). Transplanted cells were easily distinguished by fluorescent beads or by beta-galactosidase-positive expression and were distributed throughout the regenerating periodontal ligament (PL) and alveolar bone. At 1 week after wounding, animals treated with beta-galactosidase-positive cells exhibited a slightly higher percentage of labeled cells in the PL compared with the fluorescent bead-labeled cell implant group (2% vs. 1% respectively; P > 0.2). At Week 2 percentages of labeled cells were slightly increased in the regenerating PL (approximately 3% for both groups, P > 0.2). In regenerating alveolar bone at 1 week, animals that were treated with beta-galactosidase-positive cells and fluorescent bead-loaded cells exhibited approximately 30% and 25% of labeled cells respectively. At 2 weeks after wounding there was an increase in the percentage of transplanted beta-galactosidase-positive cells (approximately 39% at week 2; P < 0.05), but not of transplanted cells with fluorescent beads (approximately 25% at week 2). In sites with transplanted cells there were higher percentages of OPN positive and BSP positive cells in nascent bone and more newly formed bone than in controls (>40%; P < 0.05). Transplantation of beta-galactosidase-positive cells or cells loaded with fluorescent beads is a useful method for assessing the fate and differentiation of periodontal cells in vivo. Fluorescent beads, however, are diluted at mitosis and this method underestimates the percentage of transplanted cells. As transplanted periodontal cells in both groups promoted regeneration of alveolar bone, cell transplantation could improve the restoration of periodontium destroyed by periodontitis.

Etidronate (HEBP) promotes osteoblast differentiation and wound closure in rat calvaria.

The mechanisms that regulate the migration, proliferation and differentiation of osteogenic cell populations in vivo are poorly understood. Elucidation of these mechanisms is essential for an understanding of the basic processes that determine mineralized connective tissue homeostasis and regeneration. Bisphosphonates are known to regulate bone metabolism, in part through effects on osteoclastic resorption. Given previous data from other in vitro and in vivo investigations, we considered that they could also affect the proliferation and differentiation of osteoblasts in vivo. We tested this hypothesis using a bisphosphonate (ethane-1-hydroxy-1,1-bisphosphonate, HEBP, etidronate) and a calvarial wound model in which osteogenic differentiation and bone formation are coordinately induced by the wounding stimulus. Wounds through the calvarial bone were created in 20 male Wistar rats. After surgery, animals were treated every day for 1 or 2 weeks with HEBP or saline (controls) and five rats in each group were killed at 1 or 2 weeks following surgery. Cellular proliferation and clonal growth were assessed by 3H-thymidine labeling 1 h before death followed by radioautography. Cellular differentiation of osteogenic cell populations was determined by immunohistochemical staining for osteopontin and bone sialoprotein. Von Kossa and toluidine blue staining were used for the assessment of mineralization and osteoid formation, and for morphometric analysis of wound closure. At 1 and 2 weeks after surgery HEBP promoted wound closure (> twofold greater than controls, P < 0.001) and mineralized/osteoid tissue formation in the bony compartment of the wound (> 50% higher than saline controls, P < 0.05). In HEBP-treated animals there was a > 50% increase in intracellular staining for osteopontin in the endosteum-lined spaces adjacent to the wound (P < 0.05) and increased staining for osteopontin in the nascent bone at the wound margin (> 50% greater than controls, P < 0.05). However, there were reduced cell counts and labeling indices at stromal precursor sites (65% reduction compared to controls; P < 0.01). As HEBP increased osteopontin expression and osteoid/mineralized tissue formation but reduced the proliferation of precursor cells, we conclude that in addition to blockade of bone resorption and mineralization, this drug, at doses which also reversibly inhibit mineralization, may promote osteoblast differentiation as well.

A dental implant: aluminium trioxide exhibited no effect on mouse reproductive and mutanogenic potential.

Several diseases as well as trauma can affect the composition and integrity of periodontal tissues leading eventually to the destruction of connective tissue matrix and cells, loss of attachment and resorption of alveolar bone, often followed by tooth loss. Replacement of the missing tooth could then be provided by endosseous dental implants healing in a form of osseo- or fibrosteal integration to the alveolar bone. Aluminium oxide ceramics, a form of endosseous implant, allows osseointegration type of healing and has demonstrated excellent biocompatibility. However, potential aluminium toxicity has been implicated in the pathogenesis of a number of clinical disorders and for this reason we examined the reproductive and mutagenic effect of aluminium trioxide ceramic implant in experimental mice. 720 female and 45 fertile male BALB-cAn NCR mice were included in the study. 3 experimental groups of fertile male mice (15 for each group) were treated with an intraperitoneal injection of aluminium trioxide (1 g/ kg of body weight, group I), with ethyl-methane-sulphonate as a positive control (200 mg/kg, group II) and with Tween-80 (10 mg/kg as a negative control, Group III). Each of the labeled male mice fertilized previously uncoupled female mice during 8 weeks (a pair per week) to facilitate appropriate pre- and post-meiotic conditions of spermatogenesis to occur. Female mice were sacrificed with cervical dislocation at day 13 after fertilization. Immediately upon sacrifice the uterus was removed and the number of alive and healthy, or alive but mutated and/or dead embryos was computed to determine the dominant lethal or mutagenic effect. Animals treated with aluminium trioxide demonstrated similar effects on the reproductive and mutagenic capacity as the negative control, whereas the animals treated as positive controls exhibited significantly reduced reproductive and mutagenic capacity. Collectively, we concluded that aluminium trioxide has a very low rate of embryonal mortality and mutagenicity in mice. This finding is in general agreement with the biocompatibility of aluminium trioxide as an implant material.

The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation.

Viable periodontal ligament (PL) cells are required for PL healing of avulsed teeth following replantation. If immediate replantation cannot be accomplished, the ability of PL progenitor cells to reproduce (clonogenic capacity) and recolonize the wound may be extended by prevention of desiccation and storage in physiological media. This investigation examined the effects of storage in saliva, milk, Hank's balanced salt solution (HBSS) and Eagle's medium (alpha MEM) on the clonogenic capacity of human PL progenitor cells at 30 and 60 min extra-alveolar time. Twenty erupted human premolar teeth extracted as atraumatically as possible for orthodontic purposes were used in the present study. Fifteen premolars were placed immediately in freshly collected autologous saliva at room temperature, (+ 23 degrees C) for 15 min. These 15 premolars were next divided into three groups of five and stored in either saliva, milk or HBSS at + 4 degrees C in plastic cups surrounded by ice. The remaining five teeth served as positive controls and were immediately placed in alpha MEM at + 4 degrees C. PL tissue was scraped from one-half of the root surface with a scalpel at 30 and 60 min total extra-alveolar duration. Cells were released from the tissue sample with a 30 min enzymatic digestion procedure and the cells from the tissue samples analyzed for clonogenic capacity. There was a reduction in clonogenic capacity with time for all protocols. Periodontal ligament cells stored in alpha MEM showed the least reduction between 30 and 60 min and the greatest reduction was observed for PL cells stored in saliva. The difference in clonogenic capacity following transfer from saliva to milk or HBSS was not significant at 30 min. At 60 min, cells transferred from saliva to HBSS had a statistically higher percentage of clonogenic cells than those transferred to milk (5.9% vs. 3.5%; P < 0.05). We conclude that immediate storage of avulsed teeth in autologous saliva, followed by transfer to chilled milk, preserves the presence of sufficient progenitor cells in the PL to warrant replantation and the possibility of PL healing at 60 min extra-alveolar duration.

Osteogenic inhibition by rat periodontal ligament cells: modulation of bone morphogenic protein-7 activity in vivo.

Periodontal ligament width is precisely maintained throughout the lifetime of adult mammals but the biological mechanisms that inhibit ingrowth of bone into this soft connective tissue are unknown. As bone morphogenic proteins strongly stimulate osteogenesis and can induce ectopic bone formation in vivo, we tested the hypothesis that topical application of this powerful osteogenic agent will overwhelm the osteogenic inhibitory mechanisms of periodontal ligament cells and induce ankylosis. Wounds through the alveolar bone and periodontal ligament were created in 45 male Wistar rats. Defects were filled with either a collagen implant or collagen plus bone morphogenic protein (BMP-7), or were left unfilled (controls). Three animals per time period were killed on days 2, 5, 10, 21 and 60 after surgery for each wound type. Cellular proliferation and clonal growth in periodontal tissues were assessed by 3H-thymidine labeling 1 h before death, followed by radioautography. Cellular differentiation of soft and mineralizing connective tissue cell populations was determined by immunohistochemical staining of alpha-smooth muscle actin, osteopontin and bone sialoprotein. In regenerating periodontium, BMP-7 induced abundant bone formation by 21 days (2.5-fold greater than controls or collagen implant only; P<0.001), but by day 60 the volume of the newly formed bone had returned to baseline levels and was similar for all groups. Independent of the type of treatment, periodontal ligament width was unchanged throughout the experimental period (P>0.05). Animals treated with BMP-7 implants showed greatly increased cellular proliferation in the periodontal ligament adjacent to the wound site and in the regenerating alveolar bone at days 5 and 10 after wounding compared to the other treatment groups (P<0.005). Animals in the BMP-7 group exhibited similar spatial and temporal staining patterns for alpha-smooth muscle actin, osteopontin and bone sialoprotein as controls. Collectively, these data show that BMP-7 promoted the proliferation of precursor cells in the periodontal ligament but did not induce osteogenic differentiation in this compartment. Consequently a powerful osteogenic stimulus like BMP-7 cannot significantly perturb the mechanisms that regulate periodontal ligament width and maintain periodontal homeostasis.

Is fibroblast heterogeneity relevant to the health, diseases, and treatments of periodontal tissues?

There are wide variations of gene expression and strikingly different responses to extracellular signals among different fibroblast populations. This has prompted a large number of in vitro studies which suggest that fibroblasts are not homogeneous but instead comprise multiple subpopulations with extensive site-to-site and intra-site variations. Conceivably, either fibroblasts are not all created equal, or, alternatively, discrete subpopulations may emerge in development, inflammatory lesions, or wound healing. While the heterogeneous nature of cultured fibroblasts has been known for some time, are these variations relevant to our understanding of the biology of oral tissues, their involvement in disease, and their response to therapy? Since fibroblasts are the predominant cell type in soft connective tissue matrices, the regulation of their proliferative, synthetic, and degradative behavior is likely to be important in tissue physiology and pathology. In this review, we use the current literature to assess whether fibroblast subpopulations really make a difference in the health and disease of periodontal tissues. We address the following questions: (1) Is fibroblast heterogeneity a real in vivo phenomenon? (2) How can we advance our knowledge of phenotypic variations and the regulation of fibroblast differentiation? (3) Could a knowledge of fibroblast heterogeneity have an impact on the development of new approaches to pathogenesis and the treatment of periodontal tissues?