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PubMed2001

The effects of platelet-derived growth factor-BB on periodontal cells in an in vitro wound model.

BACKGROUND: Platelet-derived growth factor (PDGF-BB) has been shown to enhance periodontal regeneration. Principles of guided tissue regeneration dictate that one of the goals of therapy is to modulate the wound healing processes to favor repopulation of the wound with cells derived from the periodontal ligament rather than from the gingival tissues. Using an in vitro wound model, gingival fibroblasts (GF) have been shown to fill a wound space significantly faster than periodontal ligament cells (PDL). There are no data reported directly comparing the response of these 2 cell types to PDGF-BB within such a wound model. Therefore, the aims of this research were: 1) to characterize both the proliferative and wound fill (WF) effects of PDGF-BB within an in vitro model and 2) to compare specific growth factor effects between GF and PDL. METHODS: Primary cultures of both human PDL and GF were derived from explanted tissues and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer across the diameter of the wells. The wells were then incubated for 2, 6, and 9 days in media containing 0.1% fetal bovine serum (FBS) and 1 of 5 concentrations of PDGF-BB. At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU) fixed, and nuclei were stained to measure BrdU incorporation (as a measure for proliferation). Cells were counter-stained with cytoplasmic stain to measure cell number. Quantitative analyses within the wound boundaries, marginally (area of interest [AOI] 1) and centrally (AOI 2), were accomplished using computer-assisted histomorphometry. RESULTS: PDL exhibited a significantly greater proliferative response to PDGF-BB in both AOI when compared to GF (P <0.0001). The PDL exhibited increased levels of proliferation at concentrations of PDGF-BB greater than or equal to 10 ng/ml. By contrast, GF displayed no increase in proliferation in response to stimulation with PDGF-BB at any of the concentrations tested when compared to negative controls. The wound fill (WF) responses to PDGF-BB were similar between PDL and GF, with both cell types responding in an all or none fashion when measured at day 2, and in a concentration-dependent manner at later time points. The only significant difference in WF between PDL and GF occurred in AOI 2 in negative control medium (0 ng/ml of PDGF-BB), with GFs having greater (P <0.01) levels of WF over the 9 days. CONCLUSION: The findings from this study demonstrate differing effects of PDGF-BB on the proliferation of PDL and GF in this in vitro model. These results suggest that there may be cell-specific differences critical to periodontal wound healing that may be exploited in the development of new therapies.

Becaplermin↗
PubMed2002

Cytokine regulation of gingival fibroblast lysyl oxidase, collagen, and elastin.

BACKGROUND: Systemic therapy with cyclosporin A, phenytoin, and nifedipine modulates cytokine levels in human gingival tissues. Functional relationships between altered cytokine levels and gingival extracellular matrix production are partially characterized. The present study investigates in cultured human gingival fibroblasts the regulation of lysyl oxidase, alpha-1 type I collagen, and elastin by selected cytokines that are elevated in drug-induced gingival overgrowth tissues. METHODS: Normal human gingival fibroblasts were cultured and then treated with selected cytokines: interleukin (IL)-1beta, IL-6, platelet-derived growth factor (PDGF)-BB, and basic fibroblast growth factor (bFGF or FGF-2). Cells were harvested at intervals, and changes in lysyl oxidase enzyme activity, and in mRNA levels of lysyl oxidase, alpha-1 type I collagen, and elastin were determined. RESULTS: bFGF reproducibly and significantly decreased human gingival fibroblast lysyl oxidase and alpha-1 type I collagen mRNA levels in a dose- and time-dependent manner; 1 nM bFGF reduced lysyl oxidase and collagen mRNA levels to 53% and to less than 10% of control after 48 hours of treatment. Interestingly, bFGF downregulated lysyl oxidase enzyme activity by 10% to 20%. IL-1, IL-6, and PDGF-BB did not significantly regulate lysyl oxidase enzyme activity, or alpha-1 type I collagen, elastin, and lysyl oxidase mRNA levels under the conditions tested. CONCLUSIONS: Previous studies have shown that modulated levels of bFGF occur in gingiva as a result of certain pharmacologic therapies. The present study suggests that modulated levels of bFGF likely influence gingival connective tissue metabolism.

Adult↗
PubMed2003

Platelet concentrates: effects of calcium and thrombin on endothelial cell proliferation and growth factor release.

BACKGROUND: Clinical evidence suggests that platelet concentrate (PC) could have beneficial therapeutic effects on hard and soft tissue healing, due to the contents of growth factors (GFs) stored in the platelets. The objectives of this study were: 1) to determine the concentrations of platelet-derived growth factor-BB (PDGF-BB), transforming growth factor-beta1 (TGF-beta1), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) released from PCs and whole blood (WB), before and after the addition of various concentrations of calcium and thrombin, and 2) to assess the physiological importance of the released GFs on angiogenesis. METHODS: WB and PCs were harvested and prepared from three healthy volunteers. Enzyme-linked immunosorbent assay tests, specific for PDGF-BB, TGF-beta1, VEGF, and bFGF, were performed on WB and PC supernatants, collected before and 30 minutes after the addition of various concentrations of calcium and thrombin. The supernatants were also added to human umbilical vein endothelial cell (HUVEC) cultures in order to measure their effects on endothelial cell proliferation. RESULTS: Growth factor concentrations detected in PC supernatants were significantly greater (280% to 800% increase) than concentrations present in WB supernatants. Calcium and thrombin induced immediate GF release from PCs in a dose-dependent fashion. Furthermore, PC supernatants led to greater HUVEC proliferation rates than WB supernatants. However, there was no correlation between the concentrations of specific GFs and HUVEC proliferation rates. CONCLUSION: These results suggest that PCs could stimulate blood vessel formation. They also reinforce the relevance for using PCs in regenerative therapies.

Becaplermin↗
PubMed2003

In vitro evaluation of the mitogenic effect of platelet-derived growth factor-BB on human periodontal ligament cells cultured with various bone allografts.

BACKGROUND: Several studies have documented the role of growth factors in periodontal regeneration. It has been shown that platelet-derived growth factor (PDGF) is a potent stimulator of human periodontal ligament (PDL) cells. A variety of bone graft materials are used to treat osseous defects caused by periodontal disease. We evaluated the mitogenic effect of PDGF on human PDL cells cultured with different allografts to determine which of the allografts with or without PDGF promoted periodontal regeneration. METHODS: Two human demineralized freeze-dried allografts of cortical (DFDBA) and cancellous (DFBA) bone and a non-demineralized freeze-dried allograft (FBA) from cancellous bone were used alone or supplemented with PDGF-BB. Human PDL cultures were derived from the mid-root of 2 maxillary premolars extracted for orthodontic reasons. Cells were grown separately in 24-well dishes with or without 20 mg of each bone allograft. On day 2 of quiescence, new medium was added with 10 ng/ml of PDGF-BB. DNA synthesis was estimated by measuring [3H] thymidine incorporation to determine the effects of the test agents on cell proliferation. Cells were processed and subjected to scintillation counting after 48 hours of incubation. Counts per minute (cpm/well) were determined for each sample. RESULTS: There was no statistically significant difference (P<0.05) on PDL cell proliferation when the allografts were used alone. PDL cells exhibited significantly greater proliferative responses to the 2 demineralized bone allografts, DFDBA and DFBA, when combined with PDGF-BB. A statistically significant difference on DNA synthesis was noticed when PDGF-BB was added to PDL cells cultured with FBA. PDL cells displayed no significant increase in mitogenic activity when cultured with PDGF-BB alone. CONCLUSIONS: The findings of this study demonstrate the beneficial role of DFDBA, DFBA, and FBA as synergic agents with PDGF-BB to periodontal regeneration. The significant ability of the 2 decalcified bone allografts, DFDBA and DFBA, combined with PDGF to stimulate PDL cell proliferation might be a useful adjunct in the treatment of periodontal defects.

Adult↗
PubMed2003

Periodontal regeneration in humans using recombinant human platelet-derived growth factor-BB (rhPDGF-BB) and allogenic bone.

BACKGROUND: Purified recombinant human platelet-derived growth factor BB (rhPDGF-BB) is a potent wound healing growth factor and stimulator of the proliferation and recruitment of both periodontal ligament (PDL) and bone cells. The hypothesis tested in this study was that application of rhPDGF-BB incorporated in bone allograft would induce regeneration of a complete new attachment apparatus, including bone, periodontal ligament, and cementum in human interproximal intrabony defects and molar Class II furcation lesions. METHODS: Nine adult patients (15 sites) with advanced periodontitis exhibiting at least one tooth requiring extraction due to an extensive interproximal intrabony and/or molar Class II furcation defect were entered into the study. Eleven defects were randomly selected to receive rhPDGF-BB. Following full-thickness flap reflection and initial debridement, the tooth roots were notched at the apical extent of the calculus, the osseous defects were thoroughly debrided, and the tooth root(s) were planed/prepared. The osseous defects were then filled with demineralized freeze-dried bone allograft (DFDBA) saturated with one of three concentrations of rhPDGF-BB (0.5 mg/ml, 1.0 mg/ml, or 5.0 mg/ml). Concurrently, four interproximal defects were treated with a well accepted commercially available graft (anorganic bovine bone in collagen, ABB-C) and a bilayer collagen membrane. Radiographs, clinical probing depths, and attachment levels were obtained preoperatively (at baseline) and 9 months later. At 9 months postoperatively, the study tooth and surrounding tissues were removed en bloc. Clinical and radiographic data were analyzed for change from baseline by defect type and PDGF concentration. The histologic specimens were analyzed for the presence of regeneration of a complete new attachment apparatus coronal to the reference notch. RESULTS: The post-surgical wound rapidly healed and was characterized by firm, pink gingivae within 7 to 10 days of surgery. There were no unfavorable tissue reactions or other safety concerns associated with the treatments throughout the course of the study. In rhPDGF/allograft sites, the vertical probing depth (vPD) reduction for interproximal defects was 6.42 +/- 1.69 mm (mean +/- SD) and clinical attachment level (CAL) gain was 6.17 +/- 1.94 mm (both P < 0.01). Radiographic fill was 2.14 +/- 0.85 mm. Sites filled with ABB-C had a PD reduction and CAL gain of 5.75 +/- 0.5 and 5.25 +/- 1.71, respectively. Furcation defects treated with rhPDGF/allograft exhibited a mean horizontal and vertical PD reduction of 3.40 +/- 0.55 mm (P < 0.001) and 4.00 +/- 1.58 mm (P < 0.005), respectively. The CAL gain for furcation defects was 3.2 +/- 2.17 mm (P < 0.030). Histologic evaluation revealed regeneration of a complete periodontal attachment apparatus, including new cementum, PDL, and bone coronal to the root notch in four of the six interproximal defects and all evaluable (four of four) furcation defects treated with PDGF. Two of the four interproximal intrabony defects treated with ABB-C and membrane exhibited regeneration. CONCLUSIONS: Use of purified rhPDGF-BB mixed with bone allograft results in robust periodontal regeneration in both Class II furcations and interproximal intrabony defects. This is the first report of periodontal regeneration demonstrated histologically in human Class II furcation defects.

Adult↗
PubMed2003

Platelet-derived growth factor-BB stimulated cell migration mediated through p38 signal transduction pathway in periodontal cells.

BACKGROUND: Intracellular signaling pathways mediate specific responses to growth factors. The manipulation of these pathways ultimately may be used to control the clinical outcomes of periodontal regenerative therapy. The purpose of this study was to examine the role of the p38 signal transduction pathway in the responses of periodontal cells to platelet-derived growth factor-BB (PDGF). METHODS: Primary cultures of human periodontal ligament cells (PDLs) and gingival fibroblasts (GFs) were used for all experiments. Cell numbers, 3H-thymidine incorporation, and Boyden chamber assays were used to characterize the effects of SB 203580 (SB), a specific inhibitor of the p38 signaling pathway, on cell proliferation and migration. An in vitro wound model also was used to assess the effects of SB. For the in vitro wound assay, triplicate wells were incubated for 1, 3, 5, and 7 days using 0.1% fetal bovine serum (FBS), 10% FBS +/- 10 microM SB, or 20 ng/ml PDGF +/- 10 microM SB. Digital histomorphometric analysis assessed cellular fill within the wound area. RESULTS: SB specifically inhibited PDGF-induced migration in the Boyden chamber assays without affecting cell proliferation. The wound model data showed similar levels of wound fill for PDLs and GFs in 10% FBS. Relative to 10% FBS, PDLs stimulated with PDGF showed significantly (P < 0.01, analysis of variance) greater wound fill (74%) than GFs (12%). SB inhibited the PDGF-induced wound fill of PDLs and GFs by 64% and 57%, respectively. This inhibition was significant (P < 0.01, ANOVA) only for PDLs. The addition of SB to 10% FBS did not significantly affect the wound fill response of either cell type compared to 10% FBS alone. CONCLUSIONS: These results demonstrate that periodontal cells possess distinct responses to PDGF that may be altered at the signal transduction level. The manipulation of these responses through the use of inhibitors to specific signaling pathways may enhance our control of periodontal regeneration in the future.

Adult↗
PubMed2004

Lysophosphatidic acid modulates the regenerative responses of human gingival fibroblasts and enhances the actions of platelet-derived growth factor.

BACKGROUND: Platelet-derived growth factor (PDGF) has been used to promote healing in many in vitro and in vivo models of periodontal regeneration. PDGF is known to interact extensively with another platelet mediator, lysophosphatidic acid (LPA), to enhance regenerative responses in non-oral systems. PDGF and LPA are both liberated by platelets in the blood clot, which is known to be critical in stabilizing early periodontal wound healing. The purpose of this study was to evaluate the basic interactions of LPA with primary human gingival fibroblasts (GF) alone and with PDGF-BB for promoting GF growth and migration, as well as their effects in an in vitro oral wound-healing model. METHODS: GF regenerative responses were measured using 1 and 10 microM LPA in the absence or presence of 1 or 10 ng/ml PDGF-BB. Cell growth was determined using [3H]thymidine incorporation and cell counting. Migration responses were measured using a microchemotaxis chamber. For the in vitro wound-healing experiments, GF were grown to confluence on glass slides, and a 3 mm wide wound was mechanically inflicted. Percent wound fill on days 4, 6, and 9 was analyzed using computer-assisted histomorphometry. RESULTS: GF exhibited proliferative and chemotactic responses to LPA. These responses were synergistic when LPA and PDGF-BB were present together. LPA on its own did not stimulate statistically significant wound fill, but when combined with PDGF-BB, wound fill was equivalent to the 10% serum positive control group by day 6 (5.5-fold of negative control, [P<0.001]) and again on day 9 (6-fold of negative control, [P<0.001]). CONCLUSIONS: These studies provide the first evidence that LPA stimulates human GF regenerative responses and that it interacts positively with PDGF-BB to regulate these actions. The results suggest that LPA needs to be further investigated in the oral system as a factor that should be considered for incorporation when designing new periodontal wound-healing therapies using PDGF.

Becaplermin↗
PubMed2005

Repair of Class II furcation defects after a reparative tissue graft obtained from extraction sockets treated with growth factors: a histologic and histometric study in dogs.

BACKGROUND: Reparative tissue of extraction sockets was proposed as grafting material in the treatment of periodontal defects. Our hypothesis was that the addition of growth factors to extraction sockets improves the regenerative potential of this tissue when used as a graft. The objective of the present study was to analyze qualitatively and quantitatively the repair of acute Class II furcation defects after they receive this grafting material. METHODS: The second and third upper premolars were extracted from four dogs. Platelet-derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-I, at concentrations of 6 microg/ml each, were applied to the resulting sockets. After 5 days, 24 acute defects (12 control and 12 test defects) were created in the second, third, and fourth lower premolars. Only the test sites received the graft. The flaps were positioned coronally on both sides and sutured. After 45 days, the specimens were collected, decalcified, and processed histologically in a buccal-lingual plane. The parameters were measured horizontally in the buccal-lingual direction. RESULTS: Repair was histologically and histometrically similar in the two groups. No significant difference was observed between the test and control groups in the parameters connective tissue, new cementum, new bone, and junctional epithelium. CONCLUSION: The use of this graft did not show beneficial effects on the repair of acute Class II furcation defects in dogs.

Animals↗
PubMed2005

Platelet-derived growth factor enhancement of two alloplastic bone matrices.

BACKGROUND: The use of alloplastic matrices that mimic the mineral phase of bone has become a viable alternative to current mainstream therapies in dentistry such as allografts and autogenous grafts. Because alloplastic bone substitutes generally have relatively poor osteogenic properties, analyzing their potential as vehicles to deliver growth factors is an important step in assessing methods to enhance their clinical efficacy. The aim of these studies was to treat beta-tricalcium phosphate (beta-TCP) and calcium sulfate (CaSO(4)) with platelet-derived growth factor (PDGF)-BB to enhance the osteogenic capabilities of these materials. METHODS: In the beta-TCP studies, PDGF-BB adsorption and release were accomplished using (125)I radiolabeled growth factor and non-radioactive human recombinant PDGF at a ratio of 1:300 M. For the adsorption studies, the radiolabeled PDGF-BB/ non-radioactive PDGF solutions with resultant PDGF concentrations of 10(7) and 10(8) M were incubated with beta-TCP from 1 to 120 minutes, and the amount of adsorbed (125)I-PDGF-BB was measured using a gamma counter. Similar adsorption studies were conducted with a 30-minute incubation of beta-TCP with various PDGF concentrations. In vitro release studies were conducted with beta-TCP to which radiolabeled PDGF had been adsorbed as above. Release studies were also conducted with CaSO(4) that was hydrated with the radioactive PDGF solution described above for the TCP studies. In vivo PDGF-BB release from beta-TCP and CaSO(4) was evaluated in a mouse model, where the radioactive PDGF/non-radioactive PDGF-BB treated beta-TCP or CaSO(4) sample was inserted subcutaneously and later removed for radioactive measurement. Proliferation of human osteoblastic cells in the presence of PDGF- treated beta-TCP or CaSO(4) was assessed by (3)H thymidine incorporation. RESULTS: The absorption studies revealed that PDGF-BB was absorbed in a concentration and time-dependent manner to beta-TCP. In the in vitro release studies, approximately 45% of the adsorbed PDGF-BB was released after 10 days. In vivo release from both materials occurred faster than in vitro release. Osteoblastic cells incubated with PDGF-BB-treated matrices showed significantly (P <0.05, ANOVA) greater proliferation than with control matrices alone. CONCLUSION: These experiments demonstrate the feasibility of using PDGF-BB in combination with alloplastic materials such as beta-TCP or CaSO(4) to serve as more effective bone graft materials with enhanced osteogenic properties.

Adolescent↗
PubMed2005

Platelet-derived growth factor stimulates bone fill and rate of attachment level gain: results of a large multicenter randomized controlled trial.

BACKGROUND: Growth factors are generally accepted to be essential mediators of tissue repair via well-established mechanisms of action that include stimulatory effects on angiogenesis and cellular proliferation, ingrowth, differentiation, and matrix biosynthesis. The aim of this study was to evaluate in a large-scale, prospective, blinded, and randomized controlled clinical trial the safety and effectiveness of purified recombinant human platelet-derived growth factor (rhPDGF-BB) mixed with a synthetic beta-tricalcium phosphate (beta-TCP) matrix for the treatment of advanced periodontal osseous defects at 6 months of healing. METHODS: Eleven clinical centers enrolled 180 subjects, each requiring surgical treatment of a 4 mm or greater intrabony periodontal defect and meeting all inclusion and exclusion criteria. Subjects were randomized into one of three treatment groups: 1) beta-TCP + 0.3 mg/ml rhPDGF-BB in buffer; 2) beta-TCP + 1.0 mg/ml rhPDGF-BB in buffer; and 3) beta-TCP + buffer (active control). Safety data were assessed by the frequency and severity of adverse events. Effectiveness measurements included clinical attachment levels (CAL) and gingival recession (GR) measured clinically and linear bone growth (LBG) and percent bone fill (% BF) as assessed radiographically by an independent centralized radiology review center. The area under the curve (AUC), an assessment of the rate of healing, was also calculated for CAL measurements. The surgeons, clinical and radiographic evaluators, patients, and study sponsor were all masked with respect to treatment groups. RESULTS: CAL gain was significantly greater at 3 months for group 1 (rhPDGF 0.3 mg/ml) compared to group 3 (beta-TCP + buffer) (3.8 versus 3.3 mm; P = 0.032), although by 6 months, this finding was not statistically significant (P = 0.11). This early acceleration of CAL gain led to group 1 exhibiting a significantly greater rate of CAL gain between baseline and 6 months than group 3 as assessed by the AUC (68.4- versus 60.1-mm weeks; P = 0.033). rhPDGF (0.3 mg/ml)-treated sites also had significantly greater linear bone gain (2.6 versus 0.9 mm, respectively; P < 0.001) and percent defect fill (57% versus 18%, respectively; P < 0.001) than the sites receiving the bone substitute with buffer at 6 months. There was less GR at 3 months in group 1 compared to group 3 (P = 0.04); at 6 months, GR for group 1 remained unchanged, whereas there was a slight gain in gingival height for group 3 resulting in comparable GR. There were no serious adverse events attributable to any of the treatments. CONCLUSIONS: To our knowledge, this study is the largest prospective, randomized, triple-blinded, and controlled pivotal clinical trial reported to date assessing a putative periodontal regenerative and wound healing therapy. The study demonstrated that the use of rhPDGF-BB was safe and effective in the treatment of periodontal osseous defects. Treatment with rhPDGF-BB stimulated a significant increase in the rate of CAL gain, reduced gingival recession at 3 months post-surgery, and improved bone fill as compared to a beta-TCP bone substitute at 6 months.

Adult↗
PubMed2005

Effect of biologic mediators on ciprofloxacin accumulation by gingival fibroblasts.

BACKGROUND: Human gingival fibroblasts actively accumulate fluoroquinolone antimicrobials. Because fibroblasts are prevalent in gingiva, they may help sustain therapeutic fluoroquinolone levels at that site. The purpose of this study was to determine whether mediators associated with infection or injury can enhance ciprofloxacin accumulation by gingival fibroblasts. METHODS: Quiescent fibroblast monolayers were treated for 1, 6, or 24 hours with several concentrations of tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF)-2, or insulin-like growth factor (IGF)-1. Transport was assayed by measuring cell-associated fluoroquinolone fluorescence. RESULTS: All mediators significantly enhanced ciprofloxacin transport in a dose dependent manner (P < 0.05; ANOVA). Except for TNF, this enhancement was associated with a decrease in the Km of ciprofloxacin transport. Maximal enhancement was observed with 10 ng/ml PDGF or FGF and 30 ng/ml TNF, TGF, or IGF. Brief (1 hour) treatment with TNF or FGF upregulated ciprofloxacin accumulation by a maximum of 13% to 14%, whereas TGF, PDGF, and IGF enhanced this process by 19% to 24%. All of the mediators enhanced ciprofloxacin accumulation by a maximum of 19% to 24% after 6 hours and 30% to 38% after 24 hours. The accumulation of other fluoroquinolones (e.g., gatifloxacin) was also slightly enhanced. CONCLUSIONS: Gingival fibroblasts treated with cytokines or growth factors accumulate significantly more ciprofloxacin than untreated controls. This provides a mechanism by which ciprofloxacin could be preferentially distributed to gingival wound or inflammatory sites, yielding local therapeutic levels that are more sustained than in serum.

Adult↗
PubMed2006

Human periodontal fibroblast response to enamel matrix derivative, amelogenin, and platelet-derived growth factor-BB.

BACKGROUND: The ideal goal of clinical therapy in periodontal defects is regeneration of all lost structures. For regeneration to occur, cell proliferation, migration, and extracellular matrix synthesis are prerequisites. Attempts at regeneration of periodontal defects by guided tissue regeneration using bone grafts and membranes have not always yielded predictable results. Recently, attempts at engineering the defects using various materials have shown promising results. Two such approaches have been used to regenerate periodontal defects, one using extracellular matrix such as enamel matrix proteins and the other using growth factors. However, to our knowledge, no study has looked at combining these two approaches to achieve potentially even greater regeneration. METHODS: Primary human periodontal ligament (PDL) fibroblasts were explanted, and alkaline phosphatase (ALK PHOS) activity was determined. Phenotypically different cell lines were incubated for 1, 3, 6, and 10 days in 0.2% fetal bovine serum (FBS) media containing different concentrations of either enamel matrix derivative (EMD), amelogenin, platelet-derived growth factor-BB (PDGF-BB), EMD+PDGF-BB, or amelogenin+PDGF-BB. A culture of 0.2% FBS alone served as a negative control, and a culture of 10% FBS served as a positive control. Cell proliferation was measured using a Coulter counter to determine the cell number. The effects on a wound-fill model were evaluated by scraping a 3-mm wide cell-free zone in PDL monolayers across the diameter of the tissue-culture plate and determining PDL cell migration into the cell-free zone using computer assisted histomorphometry. RESULTS: Compared to the control, only EMD+PDGF-BB significantly increased PDL cell proliferation in an ALK PHOS (-) cell line (P<0.001), and EMD alone, EMD+PDGF-BB, and amelogenin+PDGF-BB significantly increased PDL cell proliferation in an ALK PHOS (+) cell line (P<0.001) with EMD+PDGF-BB showing a trend for greater proliferation than either PDGF or EMD alone. Individually, EMD and amelogenin had no significant effect on PDL cell proliferation. In the wound-fill experiment, all factors and their combinations except amelogenin significantly enhanced cell migration compared to the control (P<0.05) at the wound edge. In addition, EMD+PDGF-BB had additive effects on the ALK PHOS (-) cell line at the wound edge. At the center of the wound, neither EMD nor amelogenin had a significant wound-fill effect. However, the combination of EMD+PDGF-BB additively increased wound fill for both ALK PHOS (+) and ALK PHOS (-) cells. CONCLUSIONS: The combination of EMD and PDGF-BB produces greater proliferative and wound-fill effects on PDL cells than each by themselves. If these combined effects can be translated clinically, one may see greater regeneration in periodontal defects with this combination. However, amelogenin does not have significant effects on PDL cell proliferation or migration by itself. This may suggest that either another enamel matrix component in EMD may be responsible for some of its clinical effects, or that amelogenin alone may not trigger the regenerative potential of periodontal tissues and that it requires a combined interaction with other enamel matrix components of EMD to direct the regenerative process.

Adult↗
PubMed2006

New composite endpoints to assess efficacy in periodontal therapy clinical trials.

BACKGROUND: Clinical attachment level (CAL) and bone height (radiographic or clinical) are two well-accepted endpoint measures for periodontal clinical trials; however, neither one has been shown to be more predictive of long-term success than the other. We propose using a composite endpoint analysis combining clinical and radiological parameters to assess the beneficial effects on both hard and soft tissues following periodontal therapy using a single statistical test. To address this need, two composite endpoint alternatives are offered as a yardstick for clinical success; each includes the improvement in CAL and either improvement in linear bone growth or percent bone fill. METHODS: The data for composite endpoint analyses were derived from a clinical trial evaluating two concentrations of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) with beta-tricalcium phosphate (beta-TCP) compared to beta-TCP plus buffer as follows: group I, beta-TCP + 0.3 mg/ml rhPDGF-BB; group II, beta-TCP + 1.0 mg/ml rhPDGF-BB; and group III, beta-TCP + buffer. The construction of composite endpoints was based on the greatest values for change, accepted by the U.S. Food and Drug Administration (FDA), for clinical attachment level (DeltaCAL), mean change in radiographic linear bone gain (LBG), and mean radiographic percent bone fill (%BF), with the following dual standards defining a successful clinical result: CAL gain > or =2.67 mm and radiographic LBG > or =1.1 mm at 6 months and CAL gain > or =2.67 mm and radiographic %BF > or =14.1% at 6 months. RESULTS: Group I (beta-TCP + 0.3 mg/ml rhPDGF-BB) demonstrated statistically significant differences from group III (active control) for both composite endpoints. For the CAL/LBG composite endpoint, 61.7% of sites in group I versus 30.4% of sites in group III met the composite endpoint benchmarks (P <0.001). For the CAL/%BF composite endpoint, 70% of sites in group I versus 44.6% of sites in group III met the composite endpoint benchmarks (P = 0.003). A non-significant trend was observed for group II versus group III with 37.9% (P = 0.20) and 55.2% (P = 0.13) of sites meeting the CAL/LBG and CAL/%BF composite endpoints, respectively. These results are further emphasized by findings demonstrating a low correlation between the individual efficacy endpoints (DeltaCAL and %BF; DeltaCAL and LBG) for each of the three treatment groups. CONCLUSIONS: Composite endpoints are advantageous in periodontal clinical trials where no single efficacy endpoint has been established as the most important. A composite endpoint, combining outcome measures of both hard and soft tissue components of the periodontium, may be preferable for assessing efficacy of periodontal regenerative therapies. Two composite endpoints are offered to meet this need.

Alveolar Bone Loss↗
PubMed2003

Effect of dexamethasone on cultured human tenocytes and its reversibility by platelet-derived growth factor.

BACKGROUND: Many cases of tendon rupture after glucocorticoid injections have been reported in the literature. Despite previous studies on the histological and biomechanical changes in tendons after glucocorticoid injections, the role of glucocorticoid in causing tendon rupture still remains controversial. The objective of this study was to determine whether glucocorticoid has deleterious effects on the cellular metabolism and collagen production of cultured human tenocytes and the reversibility of these effects by platelet-derived growth factor-BB (PDGFBB). METHODS: Primary cultures of human tenocytes obtained from explants of healthy patellar tendon, harvested during anterior cruciate ligament reconstructions, were performed. The effects on cell viability, cell proliferation, and induction of apoptosis were measured by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, 5-bromo-deoxyuridine incorporation, and DNA fragmentation assay, respectively. The effect on collagen synthesis was measured by (3) H-proline incorporation assay. RESULTS: The number of viable cells was decreased, in a dose-dependent manner, by the administration of 10 (-9) to 10 (-4) -M dexamethasone. This dose range also suppressed cell proliferation. No apoptotic effect was detected. Treatment with 10 (-6) -M dexamethasone significantly reduced the amount of collagen synthesis. Co-incubation with 10 ng/mL of PDGFBB significantly reversed the effects caused by 10 (-6) -M dexamethasone. CONCLUSIONS: Dexamethasone significantly decreased cell viability, suppressed cell proliferation, and reduced collagen synthesis in cultured human tenocytes. The effects were reversed by the simultaneous administration of PDGFBB.

Anti-Inflammatory Agents↗
PubMed2005

Platelet-derived growth factor inhibits demineralized bone matrix-induced intramuscular cartilage and bone formation. A study of immunocompromised mice.

BACKGROUND: Platelet-derived growth factor (PDGF) has been proposed as a therapeutic agent to promote bone-healing. The purpose of this study was to examine the effect of PDGF on the ability of human demineralized bone matrix to induce bone formation in a nude-mouse muscle-implantation model. We also examined whether platelet-rich plasma, which contains PDGF, also modulates osteoinduction in this model. METHODS: Human demineralized bone matrix, previously shown to be osteoinductive in the calf muscles of nude mice, was mixed with PDGF-BB (0, 0.1, 1, and 10 microg/10 mg of demineralized bone matrix) and was implanted bilaterally in the calf muscles of immunocompromised (nu/nu) mice (six mice in each group). Heat-inactivated demineralized bone matrix was used as a control. Tissue was harvested at fourteen, twenty-eight, and fifty-six days after implantation. Platelet-rich plasma was prepared from the blood of a healthy donor with use of the Harvest PRP preparation device, activated with thrombin, and mixed with active and inactive demineralized bone matrix. Fifty-six days post-implantation, tissues were harvested. Osteoinduction was assessed with use of a qualitative scoring system and with quantitative histomorphometry. RESULTS: Cartilage was present at fourteen days in all tissues that had received an implant, but the amount decreased as the PDGF concentration increased. PDGF reduced bone formation at twenty-eight days in a dose-dependent manner. This inhibitory effect was resolved by fifty-six days, except in tissues in which demineralized bone matrix and 10 microg of PDGF had been implanted. In sites treated with 10 microg of PDGF, the area of new bone was decreased and the area of bone marrow was reduced at twenty-eight and fifty-six days. PDGF also appeared to retard resorption of demineralized bone matrix in a dose-dependent manner. Platelet-rich plasma reduced osteoinduction by human demineralized bone matrix that had high osteoinductive activity and had no effect on osteoinduction by demineralized bone matrix with low activity. CONCLUSIONS: PDGF inhibits, in a dose-dependent manner, intramuscular osteoinduction and chondrogenesis by demineralized bone matrix in immunocompromised mice. Platelet-rich plasma also reduces the osteoinductivity of active demineralized bone matrix.

Analysis of Variance↗
PubMed2001

Aptamers as reagents for high-throughput screening.

The identification of new drug candidates from chemical libraries is a major component of discovery research in many pharmaceutical companies. Given the large size of many conventional and combinatorial libraries and the rapid increase in the number of possible therapeutic targets, the speed with which efficient high-throughput screening (HTS) assays can be developed can be a rate-limiting step in the discovery process. We show here that aptamers, nucleic acids that bind other molecules with high affinity, can be used as versatile reagents in competition binding HTS assays to identify and optimize small-molecule ligands to protein targets. To illustrate this application, we have used labeled aptamers to platelet-derived growth factor B-chain and wheat germ agglutinin to screen two sets of potential small-molecule ligands. In both cases, binding affinities of all ligands tested (small molecules and aptamers) were strongly correlated with their inhibitory potencies in functional assays. The major advantages of using aptamers in HTS assays are speed of aptamer identification, high affinity of aptamers for protein targets, relatively large aptamer-protein interaction surfaces, and compatibility with various labeling/detection strategies. Aptamers may be particularly useful in HTS assays with protein targets that have no known binding partners such as orphan receptors. Since aptamers that bind to proteins are often specific and potent antagonists of protein function, the use of aptamers for target validation can be coupled with their subsequent use in HTS.

Animals↗
PubMed1996

Enhanced expression of platelet-derived growth factor-beta receptor by high glucose. Involvement of platelet-derived growth factor in diabetic angiopathy.

Coronary heart disease is a major complication of diabetic subjects, and platelet-derived growth factor (PDGF) has been implicated in the development of atherosclerosis. We investigated the effects of high glucose on expression of PDGF-beta receptor. In a binding assay with 125I-labeled PDGF-BB homodimer, high concentrations of glucose increased high-affinity binding of PDGF-BB on human monocyte-derived macrophages and rabbit aortic medial smooth muscle cells. Northern blot analysis confirmed the enhanced effect of glucose on expression of PDGF-beta receptor mRNA in human monocyte-derived macrophages. The protein kinase C inhibitor, staurosporin, completely suppressed an increase in PDGF-BB binding by high glucose, and high glucose significantly activated protein kinase C. These results indicated that PDGF-beta receptor expression was enhanced by high glucose through the activation of protein kinase C. Furthermore, we observed similar effects of high glucose on both PDGF-beta receptor expression and protein kinase C activation in rat mesangial cells and human capillary endothelial cells. Our results suggest that stimulation of the PDGF system is significantly involved in the development not only of diabetic atherosclerosis but also of microangiopathy.

Alkaloids↗