Sandu Pitaru

Basic fibroblast growth factor enhances the growth and expression of the osteogenic phenotype of dexamethasone-treated human bone marrow-derived bone-like cells in culture

Basic fibroblast growth factor (bFGF) was shown to enhance rat stromal bone marrow cells in culture to produce mineralized bone-like tissue in response to dexamethasone (Dex) treatment (Pitaru et al., J Bone Miner Res 8:919; 1993). The purpose of this study was to explore the effect of bFGF on Dex-treated human stromal bone marrow cells (hSBMC) in culture. Human SBMC from 6 patients were cultured for 14 days (P0) and then subcultured and grown for 28 days in the presence of Dex (10(-8) mol/L). The effect of bFGF on cell proliferation at P0 and protein content, DNA content, alkaline phosphatase activity (ALP), osteocalcin secretion, and formation of mineralized bone-like tissue (MBT) at P1 was analyzed. bFGF treatment resulted in a 2.4-fold increase in cell number at P0 and a concentration-dependent increase in [3H]-thymidine incorporation at P1, reaching a maximum increase of 3.7-fold at a concentration of 0.3 ng/mL. Furthermore, bFGF significantly increased both DNA content (two- to threefold), protein content (five- to sixfold), and the amount of MBT (up to 20-fold) at P1 cultures. Morphological evaluation of the MBT at the electron microscope level revealed a mineralization process along collagen fibrils similar to the natural process. The osteogenic nature of the bFGF-treated cultures was further shown by their ALP activity, as well as osteocalcin secretion in response to 1,25-dihydroxyvitamin D3. In conclusion, bFGF demonstrated a stimulatory effect on the proliferation of Dex-treated hSBMC-derived osteoprogenitors while maintaining their capacity to fully differentiate and form bone-like tissue in culture.

A collagenous cementum-derived attachment protein is a marker for progenitors of the mineralized tissue-forming cell lineage of the periodontal ligament

The periodontal ligament (PDL) is a fibrous and cellular connective tissue that mediates tooth attachment to bone, and it comprises fibroblastic and mineralized tissue‐forming (MTF) progenitors. The MTF progenitors are believed to give rise to the cementoblastic and osteoblastic lineages. Cementum attachment protein (CAP) is a collagenous cementum‐derived protein which binds strongly to osteoblasts, moderately to PDL cells, and weakly to gingival fibroblasts. The aim of the present study was to determine the relationship between the capacity of PDL progenitors to bind CAP and their potential to express alkaline phosphatase (ALP) and form mineralized‐like tissue in culture. Cloned human PDL progenitor populations obtained from nine human donors were assayed for their constitutive capacity to bind CAP and express ALP, and for the dexamethasone‐induced potential to form mineralized‐like tissue in culture in the presence of ascorbic acid and β‐glycerophosphate. Forty percent of the progenitor clones produced mineralized‐like tissue. Two patterns of mineralization were observed: a spread and flat pattern similar to that produced by human bone cells in culture and a nodular ridge–like type resembling that formed by human cementoma‐derived cells. A direct correlation was found between the percentage of ALP positive cells in each progenitor clone and the amount of mineralized‐like tissue formed (r = 0.565). Similar correlations were found between the number of ALP positive cells and the binding capacity of each clone (r = 0.392) and between the CAP binding capacity and mineralized‐like tissue formation (r = 0.584). Multiple regression analysis indicated that the constitutive capacity of a clone to bind CAP and express ALP is directly correlated to its dexamethasone‐induced potential to form mineralized tissue (r = 0.675). These results indicate that CAP binding and ALP expression can serve as markers for the identification of MTF progenitors in the heterogeneous cultured population of the human periodontal ligament. These data show for the first time that binding capacity to extracellular components of mineralized tissues can be a marker for mineralized tissue‐forming progenitors.

Basic fibroblast growth factor enhances the capacity of bone marrow cells to form bone-like nodules in vitro

The role of basic fibroblast growth factor (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4‐fold at a concentration of 0.3 ng/ml and half‐maximal effect was observed at a concentration of 15 pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6‐fold. Continuous treatment with bFGF (for 21 days) resulted in a 6.3‐fold increase in the culture dish surface area covered by bone‐like mineralized tissue. Maximal bone‐like tissue formation was observed in the presence of 3 ng/ml bFGF with half‐maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast‐like phenotype.

The Effects of Titanium and Hydroxyapatite on Osteoblastic Expression and Proliferation in Rat Parietal Bone Cultures

Titanium and hydroxyapatite are used for the fabrication of dental and orthopedic implants. The longevity of these implants depends on the amount and rate of bone formation that occurs around their surfaces. In the present study, the effects of titanium, hydroxyapatite, and polystyrene (control) on the proliferation of rat calvarial cells, and on their capacity to express alkaline phosphatase and respond to parathyroid hormone (PTH) stimulation, were studied. The nature of the substrate did not affect the DNA and protein contents of experimental and control cultures throughout the experimental period. Alkaline phosphatase expression and PTH response, as assessed by DNA synthesis and adenylate cyclase activity, were higher in cultures grown on hydroxyapatite and polystyrene than in those grown on titanium. These results indicate that hydroxyapatite was a more favorable substrate than titanium for the growth and differentiation of osteoblast-like cells in vitro.

Bone Morphogenetic Protein 2 Induces the Expression of Cementum Attachment Protein in Human Periodontal Ligament Clones

Cementum is continuously formed during the lifetime of a tooth. The paravascular zones in the adult periodontal ligament (PL) comprise the progenitors for the fibroblastic (Fb) lineage and mineralized tissue-forming (MTF) cell lineages--the osteoblastic (Ob) and cementoblastic (Cb) lineages. Recent studies indicate that cementum attachment protein (CAP) is related to the differentiation of the Cb lineage and is instrumental in differentiating between the three periodontal cell lineages. The purpose of this study was to assess the effect of bone morphogenetic protein 2 (BMP2) on the expression of cementum attachment protein (CAP) and on the differentiation of cloned PL progenitors. The effect of BMP2 on CAP expression and on the differentiation of cloned Fb and MTF progenitors was tested by assessing the expression of alkaline phosphatase (ALP), CAP, and bone sialoprotein (BSP) by immunochemistry and by determining the CAP-binding capacity of these clones. Untreated Fb clones were negative for all tested markers and had low CAP-binding capacity. Untreated MTF clones had a high CAP-binding capacity and were positive for the three markers. BMP2 enhanced the CAP-binding potential of both Fb and MTF clones. BMP2 induced the expression of CAP, ALP, and BSP in the Fb clones and enhanced the expression of CAP and BSP in the MTF clones. These results indicate for the first time that BMP2 can recruit progenitors to the Cb lineage and regulate the differentiation of the Cb lineage by inducing and enhancing the expression of CAP, a cell lineage-specific regulator. Furthermore, the results suggest that the MTF and Fb lineages may originate from a common early progenitor cell.

Cementum Attachment Protein Enriches Putative Cementoblastic Populations on Root Surfaces in vitro

We tested the capacity of cementum attachment protein (CAP) to recruit putative cementoblastic populations to root surfaces in vitro by determining the phenotypic expression of periodontal ligament cloned cell populations. The clones were derived from cells that attached to either CAP-coated (experimental) or uncoated (control) root slices. Root slices were co-cultured with primary human periodontal ligament cells. Cloned and parent populations were analyzed for their capacity to express alkaline phosphatase (AP), osteopontin, bone sialoprotein (BSP), and CAP and to form mineralized tissue in vitro. The percentage of CAP- and BSP-positive clones was significantly higher in the experimental clones than in the controls. The percentage of cells positive for AP, BSP, and CAP was higher in the experimental clones than in their control counterparts. Mineralized tissue formation was observed only in the cell populations derived from the CAP-coated root slices. These results indicate that CAP is capable of recruiting putative cementoblastic populations on root slices in vitro and therefore might play an important role in cementogenesis during periodontal homeostasis and wound healing.

A two-stage phase I trial of Evolence30 collagen for soft-tissue contour correction.

Background: The ideal dermal filler should be nonpermanent but with a durable effect lasting between 1 and 2 years, which is not the case with the resorbable fillers that are currently available. Evolence30 is a new, porcine-derived collagen gel based on the Glymatrix cross-linking technology, which results in a more natural and longer-lasting collagen product. Methods: In this first clinical trial of Evolence30 (30 mg/ml), the safety and efficacy of this new filler were tested and compared with those of Zyplast (bovine cross-linked) collagen, after treatment of nasolabial folds in 12 volunteers. Safety assessments included two hypersensitivity tests, physical examination of injections sites, punch biopsies for histopathology, adverse events, and blood sample analysis. The seven-grade, validated Modified Fitzpatrick Wrinkle Scale was used by three independent blinded assessors to evaluate efficacy. Results: No treatment-related adverse events were reported. Only transient erythema was observed in both treated sides, and there were no abnormal laboratory findings. None of the sera contained immunoglobulin (Ig) M, IgA, or IgE antibodies against porcine collagen at any time during the study. Initially, Evolence30 and Zyplast improved wrinkle severity to a similar extent. However, in an average follow-up of 18 months, assessment by the blinded assessors showed that the treatment effect on the Evolence30-treated side was superior in 9 of the 11 participants who were treated (p = 0.022). Conclusions: Evolence30 is a new, porcine-derived collagen product based on the Glymatrix cross-linking technology that enables a safe and effective correction of the nasolabial folds. This correction lasts significantly longer than that with Zyplast.

Immunolocalization of a Human Cementoblastoma-conditioned Medium-derived Protein

Little is known about the molecular mechanisms that regulate the cementogenesis process, because specific cementum markers are not yet available. To investigate whether a cementoblastoma-conditioned medium-derived protein (CP) could be useful as a cementum biological marker, we studied its expression and distribution in human periodontal tissues, human periodontal ligament, alveolar bone, and cementoblastoma-derived cells. In human periodontal tissues, immunoreactivity to anti-CP was observed throughout the cementoid phase of acellular and cellular cementum, cementoblasts, cementocytes, cells located in the endosteal spaces of human alveolar bone, and in cells in the periodontal ligament located near the blood vessels. Immunopurified CP promoted cell attachment on human periodontal ligament, alveolar bone-derived cells, and gingival fibroblasts. A monoclonal antibody against bovine cementum attachment protein (CAP) cross-reacted with CP. These findings indicate that CP identifies potential cementoblast progenitor cells, is immunologically related to CAP species, and serves as a biological marker for cementum.

Rapid palatal expansion: Part 1. Mineralizationpattern of the midpalatal suture in cats

The mineralization pattern of the midpalatal suture after rapid palatal expansion was investigated in 10 treated and 2 control cats, in light of the tendency of RPE to relapse. The rapid palatal expansion treatment consisted of active (25 days), retention (60 days), and relapse (60 days) phases. Standardized occlusal radiographs were taken periodically and analyzed for suture width, suture optical density in anterior vs. posterior regions, and suture area measurements of radiopaque vs. radiolucent zones. Nine cats exhibited suture splitting. During the active phase, the radiolucent zone (nonmineralized tissue) increased 12-fold and the increase in optical density was 50% greater in the anterior over the posterior suture region, demonstrating increased formation of loose connective tissue at the anterior region. During the retention period, the sutures radiopaque zone (mineralized tissue) increased by 62%, the radiolucent zone declined (64%) and the suture width decreased (65%) indicating reorganization of mineralized tissue. The decrease in optical density (increased mineralization) was 2.5 times greater in the posterior over the anterior suture region, indicating that the remineralization (closure) pattern of the expanded suture is analogous to a zipper closed in a posteroanterior direction. During the relapse phase, the reduction in total suture area (41%) and in the radiopaque zone (32%) indicates medial convergence of the maxillary horizontal processes. From our findings we extrapolated that the retention of the suture anterior region should be longer than the posterior region to catch up the lag in rebuilding and maturation of the newly deposited hard tissue.

Effect of maturation on the osteogenic response of cultured stromal Bone marrow cells to basic fibroblast growth factor

Formation of bone-like tissue in culture by stromal bone marrow cells (SBMC) derived from young growing rats is dependent on dexamethasone (Dex) (Cell Tissue Res 254:317; 1988) and is significantly enhanced by basic fibroblast growth factor (bFGF) (J Bone Miner Res 8:919; 1993). The aim of this study was to examine the effect of maturation on the osteogenic potential and the response to Dex and bFGF of SBMC by using cultures derived from young growing (6 weeks old) and adult (9 months old) rats. SBMC cultures were grown in the presence of Dex (10(-8) or 10(-7) mol/L) at both P(0) and P(1) and either in the presence or absence of bFGF. The effect of Dex and bFGF on mineralized bone-like tissue (MBT) formation was assessed at P(1). The highest levels of mineralized tissue formation in P(1) subcultures in the absence of bFGF were obtained when cultures derived from young rats (6 weeks old) were treated with Dex 10(-7) and 10(-8) mol/L at P(0) and P(1), respectively, and when cultures derived from adult rats were exposed to Dex 10(-8) mol/L both at P(0) and P(1). Under these optimal Dex concentrations, the amount of MBT formed by adult rat-derived cultures was 15-fold lower than that of young rat-derived ones. The addition of bFGF to P(0) cultures or to P(1) cultures grown under optimal Dex conditions enhanced MBT formation in P(1) cultures derived from both young and adult rats, but this effect was considerably more pronounced in the adult rat-derived cultures. The maximal levels of MBT formation were produced by cultures derived from adult rats treated with bFGF at both P(0) and P(1), whereas in cultures derived from young rats, the addition of bFGF at P(0) was not necessary for maximal MBT production. This stimulating effect of bFGF on MBT formation by adult rat-derived cultures was accompanied by a 2.2-, 1.8-, and 4.3-fold increase in proliferation, alkaline phosphatase activity, and Ca(2+) deposition rate, respectively. bFGF increased the level of glucocorticoid receptor by approximately 2. 3-fold in Dex-treated cultures derived from young animals. These results indicate that maturation is associated with a decrease in the proportion of osteoprogenitor cells in the stromal bone marrow and in their capacity to express the osteogenic phenotype. They further point to the significant role of bFGF in stimulating proliferation and osteogenic expression of stromal bone marrow osteoprogenitors derived from adult rats.