Activated platelets increase fibrinolysis of mesenchymal progenitor cells.

Bone regeneration is initiated by the formation of a blood clot. Activated platelets within this fibrin-rich matrix release signaling molecules that can attract mesenchymal progenitor cells. To gain insight into the cellular mechanism by which activated platelets can support the immigration of mesenchymal progenitors, we have tested the hypothesis that platelet-released signaling molecules increase the capacity of bone marrow stromal cells (BMSC) to activate plasminogen. We report herein that platelet-released supernatants (PRS) elevate total urokinase-type plasminogen activator (uPA) and total plasminogen activator inhibitor-1 (PAI-1) levels in BMSC, as assessed by immunoassay. Quantitative polymerase chain reaction showed an upregulation of uPA, uPA receptor, and PAI-1. Zymography and kinetic analysis based on casein hydrolysis revealed enhanced activity of cell-associated uPA upon exposure of BMSC to PRS. Inhibiting c-Jun N-terminal kinase (JNK) and phosphatidylinositol 3-kinase (PI3K) signaling reduced uPA production and decreased plasminogen activation. Corresponding Western blot analysis showed increased phosphorylation of JNK and AKT in BMSC treated with PRS. These results suggest that activated platelets can enhance the plasminogen activation capacity of mesenchymal progenitors through the stimulation of uPA production, requiring JNK and PI3K/AKT signaling. By this mechanism platelets may contribute to the organization of the blood clot during bone regeneration.

Bone cell responsiveness to growth and differentiation factors under hypoxia in vitro.

PURPOSE: Osteogenic cells contribute to the process of osseointegration and graft consolidation. However, whether the cells survive low oxygen tension and maintain their responsiveness to natural and therapeutic growth and differentiation factors remains unknown. MATERIALS AND METHODS: To determine the effects of low oxygen tension on osteogenic cell viability and responsiveness in vitro, human bone cells were placed into plastic pouches intended to create anaerobic conditions and were either simultaneously or subsequently exposed to supernatants from activated platelets or recombinant bone morphogenetic protein (BMP)-6. RESULTS: Bone cells cultured for up to 72 hours under hypoxia moderately decreased their metabolic activity, which was paralleled by morphologic changes but not by cleavage of the apoptosis markers caspase-3 and poly(ADP)ribose polymerase. Hypoxia suppressed the mitogenic response of bone cells to platelet-released supernatant and the expression of osteogenic differentiation markers alkaline phosphatase and osteocalcin upon incubation with BMP-6. Stimulation of bone cells with platelet-released supernatant and BMP-6 immediately after re-establishment of normoxia caused a moderate cellular response. However, when bone cells were allowed to recover for 7 days under normoxia, their responsiveness was equal to that of cells not previously exposed to low oxygen tension. CONCLUSIONS: These findings suggest that osteogenic cells can survive transient hypoxia and retain their potential to respond to growth and differentiation factors once normoxia is re-established. The data also implicate that reoxygenation, and thus blood vessel formation, may be an important determinant for the process of osseointegration and graft consolidation.

Activated platelets positively regulate RANKL-mediated osteoclast differentiation.

Platelets induce osteoclastogenesis in total bone marrow cultures where hematopoietic cells can interact with stromal cells. Whether or not activated platelets directly act on hematopoietic cells to promote their differentiation into osteoclasts remains unknown. Here we report that platelet releasates (PRS) increase osteoclastogenesis in stroma-depleted, macrophage colony-stimulating factor (M-CSF)-dependent bone marrow cells when cultured in the presence of receptor activator of NF-kappaB ligand (RANKL). The increased number of tartrate-resistant acid phosphatase-positive multinucleated cells (MNC) was paralleled by an enhanced transcription of osteoclast specific genes. Osteoclastogenesis was observed with hematopoietic cells previously depleted of B-cells or T-cells. Immunoprecipitation of transforming growth factor-beta (TGF-beta) decreased the osteoclastogenic capacity of the PRS. PRS enhanced phosphorylation of Smad-2, a downstream signaling mediator of TGF-beta. PRS increased phosphorylation of p38 and c-Jun NH(2)-terminal kinase (JNK), whereas only blocking of p38 but not JNK signaling suppressed osteoclastogenesis. These results suggest that activated platelets can enhance osteoclastogenesis by providing a source of TGF-beta and by activating osteoclastogenic signaling pathways.

Plasminogen activation by fibroblasts from periodontal ligament and gingiva is not directly affected by chemokines in vitro.

OBJECTIVE: Chronic inflammation in periodontal disease is associated with increased plasminogen activation and elevated levels of chemokines. It is unknown whether chemokines can regulate the activation of plasminogen via modulation of plasminogen activators (PA) and the corresponding plasminogen activator inhibitors (PAI) in periodontal tissue. DESIGN: To establish a link between chemokines and activation of plasminogen, human periodontal ligament fibroblasts (PDL) and gingival fibroblasts (GF) were incubated with IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and platelet factor-4, either alone or in the presence of the inflammatory mediators TGF-beta and IL-1. The potential of the cell lysates to activate plasminogen was based on kinetic studies with the substrate casein. Casein zymography was performed to determine the molecular sizes of the PA. Total PAI-1 in the cell-conditioned medium was quantified by immunoassay. RESULTS: We report that the chemokines did not affect activation of plasminogen by PDL and GF. Even in the presence of TGF-beta which suppressed, and IL-1 which stimulated plasminogen activation, the chemokines had no direct effect. Inhibition of PA and plasmin, but not of matrix metalloproteinases and cysteine proteinases prevented caseinolysis. The plasminogen activation capacity of the cell lysates was represented by a single band with features of uPA. The immunoassay showed that the release of PAI-1 in PDL and GF remained unaffected by the chemokines, also when stimulated with TGF-beta. CONCLUSIONS: These results suggest that plasminogen activation by PDL and GF is not directly affected by the chemokines even in the presence of the inflammatory mediators TGF-beta and IL-1.

Activated platelets retain their potential to induce osteoclast-like cell formation in murine bone marrow cultures.

Supernatants immediately obtained after platelet activation can induce osteoclast-like cell formation in murine bone marrow cultures. Here we report that activated platelets retain their potential to induce osteoclast-like cell formation over a 3-day period with repeated washing, when co-cultured with murine bone marrow cells. Supernatants obtained from washed platelets 3 days following their activation with thrombin, caused the differentiation of haematopoietic progenitors into osteoclast-like cells. The platelet-derived soluble factor(s) responsible for the induction of osteoclastogenesis can be retained in an ultrafilter with a nominal molecular weight limit of 10 kDa, and loose their activity when incubated at 99 degrees C. Indomethacin, which inhibits cyclooxygenase activity, and osteoprotegerin, a decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL), suppressed the formation of osteoclast-like cells in this model. The in vitro findings presented here suggest that activated platelets can induce osteoclast-like cell formation via a prostaglandin and RANKL-dependent mechanism over a time period corresponding to the existence of a blood clot.

Age-related changes of cell outgrowth from rat calvarial and mandibular bone in vitro.

AIM: To establish whether within an autograft ageing has an impact on the number of cells capable of responding to growth and differentiation factors released at defect sites. MATERIAL AND METHODS: The number of cells that grow out from calvarial and mandibular explants of rats 6 weeks and 9 months of age was evaluated and their response to mitogenic, chemotactic, and differentiation factors was characterized in vitro. The cell number was determined by automated counting, proliferation was evaluated by measuring the amount of (tritiated) (3)[H]thymidine incorporated into the DNA, and migration was assessed with the modified Boyden chamber assay. Alkaline phosphatase activity served as a marker of osteogenic differentiation. RESULTS: A confluent layer of cells was observed in mandibular and calvarial explant cultures derived from young rats within 10 days, while only sporadic cells were counted in cultures from adult animals. The number of cells derived from calvarial and mandibular bone of young rats was nine and five times higher, respectively, than that in explants from adult rats. Cells isolated from calvarial and mandibular explants responded to platelet-released supernatant by increased proliferation and migration in both age groups. Upon incubation with bone morphogenetic protein (BMP-6), the alkaline phosphatase activity of calvaria-derived cells from both age groups was higher than in unstimulated controls. Only one sample of cells derived from mandibular explants showed responsiveness to BMP-6 in the young age group, whereas none of the cell samples responded to BMP-6 in the adult age group. CONCLUSION: The number of cells capable of growing out from mandibular and calvarial grafts in vitro is lower in adult animals than in young animals. However, bone-derived cells from both young and adult rats respond to growth and differentiation factors present at defect sites, while there are topographic differences in the responsiveness to BMP-6.

Soluble RANKL in crevicular fluid of dental implants: a pilot study.

BACKGROUND: Receptor activator of NF-kappaB ligand (RANKL), a member of the tumor necrosis factor superfamily, is a key mediator of osteoclast formation, activation, and survival. Thus, it is reasonable to hypothesize that there might be a functional relationship between RANKL expression and peri-implantitis. PURPOSE: This pilot study was performed to determine the reference levels for soluble RANKL (sRANKL) in peri-implant crevicular fluid and to correlate them with the clinical parameters associated with inflammatory reactions and bone destruction. MATERIALS AND METHODS: The clinical parameters probing depth (PD), modified bleeding index (MBI), and modified plaque index (MPI) served as indicators for bone resorption and inflammation. Exclusion criteria for calculations were the detection limit of the immunoassay and the minimum acceptable crevicular volume for measurement. From the 84 collected samples of 16 patients, 30-84 years of age, with a total of 19 implants, 29 met these criteria. The absolute amount of sRANKL within crevicular fluid adsorbed to filter strips was a median of 0.18 femtomol (fmol; range, 0.08-0.53) and 0.26 nM (range, 0.09-1.21) when normalized by volume. PD was 4 mm in median and varied within a range between 2 and 12 mm. RESULTS: Absolute amounts of sRANKL showed no correlation with the adsorbed volume and the clinical parameters PD, MBI, and MPI. When sRANKL was normalized by volume, no correlation with the clinical parameters PD, MBI, and MPI was observed either. The patients' age was not associated with total sRANKL and the concentration of RANKL within crevicular fluid. Absolute levels of sRANKL and sRANKL concentration did not show any differences based on the sampling sites buccal and lingual, or on the patients' gender. A significant difference in sRANKL concentration was detectable when samples from maxillary implants (0.31 nM median; range, 0.12-1.21) were compared with samples from mandibular implants (0.21 nM median; range, 0.09-0.6) (p=.03). Absolute levels of sRANKL were not different between the maxilla and the mandible. CONCLUSION: Given the limited sample size, our data provide a basis for future prospective longitudinal studies on the possible relevance of sRANKL as a prognostic marker in peri-implantitis, and for an understanding of the pathophysiologic process of the disease as a prerequisite for the design of treatment strategies.

Bone marrow stromal cells of young and adult rats respond similarly to platelet-released supernatant and bone morphogenetic protein-6 in vitro.

BACKGROUND: Age-related changes in periodontal bone regeneration, osseointegration of dental implants, and graft consolidation are increasingly considered in treatment planning. This study was intended to show whether aging is associated with a diminished responsiveness of osteoprogenitor cells to growth and differentiation factors. METHODS: We compared the capacity of bone marrow stromal cells harvested from young and adult rats to proliferate, migrate, and differentiate into the osteogenic lineage following exposure to platelet-released supernatant (PRS) or bone morphogenetic protein-6 (BMP-6). Bone marrow stromal cells were isolated from 12 young rats aged 6 weeks and 12 adult rats aged 9 months. Proliferation was assessed by 3[H]thymidine incorporation, migration was evaluated with the Boyden chamber assay, and osteogenic differentiation was deduced from alkaline phosphatase activity. RESULTS: Irrespective of the donor age, bone marrow stromal cells showed increased mitogenic activity and chemotactic motility when exposed to PRS. Adult bone marrow stromal cells had higher alkaline phosphatase activities at baseline and upon incubation with BMP-6 than cells obtained from young animals. There was no difference between the two groups in the slope of the alkaline phosphatase activity curve following stimulation with BMP-6. CONCLUSIONS: The data demonstrate that, irrespective of their age, bone marrow stromal cells respond similarly to PRS and BMP-6 under in vitro conditions. These findings suggest that osteoprogenitor cells within the bone marrow of adult rats retain their juvenile potential to respond to growth and differentiation factors, which are released naturally or are applied therapeutically at sites of bone regeneration.

Osteogenic differentiation induced by bone morphogenetic proteins can be suppressed by platelet-released supernatant in vitro.

Both bone morphogenetic proteins (BMPs) and growth factors released from activated platelets both occur at sites of bone regeneration but their functional relationship to regulate the temporal and spatial sequence of cellular events is not well defined. Here we investigated whether supernatants derived from activated platelets can modulate the response of the osteogenic cell line MC3T3-E1 to BMPs, and whether BMPs have an effect on MC3T3-E1 cells stimulated with platelet-released supernatant. Platelet-released supernatant suppressed BMP-2-, BMP-6-, and BMP-7-induced osteogenic differentiation of MC3T3-E1 cells, as indicated by the significant decrease in alkaline phosphatase activity and lower levels of osteocalcin transcripts, whereas BMP-2, BMP-6, and BMP-7 did not modulate migration and proliferation of MC3T3-E1 cells, which were stimulated with platelet-released supernatant. Osteogenic differentiation in response to BMPs was not affected after precultivation of MC3T3-E1 cells with platelet-released supernatant. These data suggest that activated platelets can provide a microenvironment that temporarily suppresses the differentiation of osteogenic cells in the presence of BMPs. Future strategies to stimulate bone regeneration should take the suppression of BMP-induced osteogenic differentiation during the existence of the blood clot into account.

Cathepsin K levels in the crevicular fluid of dental implants: a pilot study.

OBJECTIVE: To determine the concentration of cathepsin K secreted into the crevicular fluid around dental implants and its correlation with clinical parameters of healthy implants and implants showing clinical signs of peri-implantitis. MATERIAL AND METHODS: Nineteen patients with 40 implants with and without peri-implantitis were enrolled in the study. Peri-implantitis was diagnosed by the pocket probing depth (PD), the modified bleeding index (MBI), the modified plaque index (MPI) and by radiographic signs of bone loss. Gingival crevicular fluid collected from the buccal and lingual sites was adsorbed to filter strips. Cathepsin K levels and total protein within the crevicular fluid were determined by immunoassay and the bicinchoninic method, respectively. RESULTS: Cathepsin K per filter strip normalized to the time of collection was 10.1 (0-33.5) pmol/sample around control implants and 22.4 (3.7-56.3) pmol/sample in the peri-implantitis group. The difference between the medians was significant (p < 0.01). Absolute cathepsin K levels in the crevicular fluid of all implants investigated showed a positive correlation with PD (R = 0.25; p = 0.03), MPI (R = 0.28; p = 0.01) and MBI (R = 0.32; p < 0.01). Absolute cathepsin K levels in the crevicular fluid also correlated with the adsorbed volume of gingival crevicular fluid (R = 0.51; p < 0.01). When normalized to the adsorbed volume of gingival crevicular fluid, the concentration of cathepsin K was 2.2 (0.01-6.4) nM around control implants and 1.7 (0.4-4.6) nM in the peri-implantitis group (p = 0.33). Patients' age correlated with sample volume and with cathepsin K normalized to the adsorbed volume of gingival crevicular fluid (R = 0.39; p < 0.01). Moreover, significant differences between male and female (p < 0.01, p < 0.01), and between mandible and maxilla (p < 0.05, p < 0.01), but not between buccal and lingual sites (p = 0.99, p= 0.93), were observed when analysed for the parameters adsorbed volume and absolute cathepsin K levels. CONCLUSION: Clinical parameters of peri-implantitis are associated with a higher amount of cathepsin K and a higher volume adsorbed to filters strips. To establish cathepsin K as a biochemical parameter to monitor peri-implant tissue health, age, sex and collection site should be considered to avoid interfering influences because of sample inhomogenity. Also a prospective study over time including more patients would be necessary.

Bone marrow stromal cells can provide a local environment that favors migration and formation of tubular structures of endothelial cells.

Findings suggest that mesenchymal progenitor cells can support the process of blood vessel formation, which may be relevant during granulation tissue formation at defect sites. The aim of this study was to investigate possible mechanisms of the angiogenic process that can be stimulated by mesenchymal progenitor cells. In the in vivo-like model of the chick embryo chorioallantoic membrane assay, we observed blood vessel ingrowth into collagen sponges containing conditioned medium from undifferentiated bone marrow stromal cells. In the Boyden chamber assay, the conditioned medium was chemotactic for human umbilical vascular endothelial cells and human uterus microvascular endothelial cells, and when cells were placed on Matrigel-coated culture dishes, formation of tubular structures was enhanced. The presence of vascular endothelial growth factor-neutralizing antibodies did not affect the outcome of the two in vitro assays. Bone marrow stromal cell-conditioned medium had no effect on proliferation of endothelial cells, as determined by measuring [3H]thymidine incorporation, and on matrix metalloproteinase 2 expression, as evaluated by reverse transcription-polymerase chain reaction and gelatin zymography. These data indicate that mesenchymal progenitor cells can provide a local environment that supports the ingrowth of blood vessels into a defect site.

Platelets can neutralize hydrogen peroxide in an acute toxicity model with cells involved in granulation tissue formation.

Platelets play a key role in the replacement of the blood clot with granulation tissue during the early steps of bone regeneration. We hypothesized that activated platelets can neutralize locally produced reactive oxygen species, thereby protecting cells involved in granulation tissue formation. The potential of platelet-released supernatant (PRS) to neutralize hydrogen peroxide (H(2)O(2)) was tested in an acute toxicity model with osteogenic, inflammatory, and endothelial cells. In the human fetal osteoblastic cell line 1.19 (hFOB), considerable morphological changes, cell shedding, and dysfunction of the respiratory chain were observed when cells were exposed to 3 mM H(2)O(2). Caspase-3 and poly-(ADP-ribose)-polymerase were not activated, suggesting that cell death occurred by necrosis. Preincubation of osteogenic cells, leukocytes, or endothelial cells with PRS decreased the acute toxicity of H(2)O(2). The capacity of platelets to release H(2)O(2)-detoxifying activity was retained for up to 72 h. Aminotriazole, an inhibitor of catalase, decreased the cytoprotective activity of PRS, whereas blocking of glutathione peroxidase by mercaptosuccinate had no effect. These results suggest that platelet-released catalase can rapidly neutralize cytotoxic amounts of H(2)O(2), a process that may play a role during the early stages of bone regeneration.

Proliferation and osteogenic differentiation of cells from cortical bone cylinders, bone particles from mill, and drilling dust.

PURPOSE: The osteogenic potential of a graft is based on the parallel lines ability of cells to survive transplantation and to respond to local factors that stimulate new bone formation. Here we investigated the potential of cells that had grown out of porcine cortical bone grafts obtained by 3 preparation techniques to respond to mitogenic and osteogenic stimuli. MATERIALS AND METHODS: Bone grafts were harvested from 2 pigs. Cortical bone was taken in cylindrical form and ground in a bone mill or harvested via drilling and aspiration. RESULTS: Cell outgrowth was observed in all cortical bone cylinders, bone cylinders homogenized by mill, and 5 of 10 explants of bone dust collected upon drilling. After a 2-week culture period, the number of outgrown cells did not significantly differ among the 3 preparations. Bone cells showed increased proliferation in response to platelet-released supernatants as determined by 3 [H]-thymidine incorporation assay. When cultured under conditions that favor the expression of an osteogenic phenotype, the outgrown cells expressed alkaline phosphatase activity and transcripts of the osteoblast-specific marker osteocalcin. Individual cell preparations showed accumulation of mineral salts in their extracellular matrix. Bone cells also increased alkaline phosphatase activity in response to bone morphogenetic protein (BMP)-2, BMP-6, and BMP-7. The mitogenic and osteogenic response was obtained with cells from the mandible as well as from the maxilla, irrespective of the preparation technique. CONCLUSION: These data show that cortical bone grafts contain cells that have the ability to proliferate and differentiate into the osteogenic lineage, suggesting that these cells can contribute to bone regeneration following transplantation.

Platelet-released supernatant increases matrix metalloproteinase-2 production, migration, proliferation, and tube formation of human umbilical vascular endothelial cells.

BACKGROUND: Local application of platelets represents a promising tool to enhance bone regeneration. New bone formation strictly requires blood vessel formation, a sequential process involving matrix degradation, migration, proliferation, and tube formation of endothelial cells. Here we investigated the impact of secreted granula products from activated platelets on endothelial cells, and determined the involvement of extracellular signal-regulated kinase (ERK) signaling. METHODS: The effects of platelet-released supernatant on endothelial cells were investigated using in vitro models. Matrix metalloproteinase-2 (MMP-2) release, migration, proliferation, and tube formation of human umbilical vascular endothelial cells (HUVEC) were determined in response to platelet-released supernatant by gelatine zymography, Boyden chamber assay, 3[H]thymidine incorporation, and basement membrane assay, respectively. All experiments were performed in the presence of the ERK signaling inhibitor PD98059. ERK phosphorylation was detected by Western blot analysis. RESULTS: Incubation with platelet-released supernatant increased the production of MMP-2, migration, proliferation, and tube formation of HUVEC. Platelet-released supernatant also stimulated ERK phosphorylation in HUVEC. Inhibition of ERK signaling decreased platelet-released supernatant-stimulated endothelial cell proliferation, but not MMP-2 activity, migration, and the formation of capillary tubes. CONCLUSIONS: Our data suggest that secreted granula products from platelets can enhance different stages of blood vessel formation, and that ERK signaling is required to mediate the mitogenic effects of the supernatant. These findings support the hypothesis of a potential link between platelet activation and blood vessel formation during bone regeneration.

Porcine sinus mucosa holds cells that respond to bone morphogenetic protein (BMP)-6 and BMP-7 with increased osteogenic differentiation in vitro.

The aim of this in vitro study was to determine whether the sinus mucosa holds cells with an osteogenic potential. Frozen sections of sinus mucosa from three adult pigs were investigated for the expression of STRO-1, a marker of mesenchymal progenitor cells, and alkaline phosphatase activity, an enzyme expressed by cells committed to the osteogenic lineage and by mature osteoblasts. To determine their osteogenic potential, mucosa-derived cells were incubated with bone morphogenetic protein (BMP)-6 and BMP-7, and alkaline phosphatase activity, osteocalcin expression, and mineralization of the extracellular matrix was measured. We found sinus mucosa cells staining positive for STRO-1 and alkaline phosphatase activity. When sinus mucosa tissue was placed in culture, alkaline phosphatase positive cells grew out from the explants and further increased alkaline phosphatase activity in response to BMP-6 and BMP-7. The expression level of the osteoblast-specific extracellular matrix protein osteocalcin, and the amount of calcium accumulation within the extracellular matrix was also increased in response to BMPs. We conclude that the sinus mucosa holds mesenchymal progenitor cells and cells committed to the osteogenic lineage that can respond to BMP-6 and BMP-7 by an increase of their osteogenic differentiation.

Platelet-released supernatants increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions.

Platelet-rich plasma is currently promoted to serve as an adjuvant for bone grafts to enhance quantity and quality of newly forming bone; however, the underlying cellular mechanisms are not fully understood. We show here that supernatants of leukocyte-depleted thrombin-activated platelets increase migration and proliferation, and decrease osteogenic differentiation of bone marrow-derived mesenchymal progenitor cells under in vitro conditions. Using neutralizing antibodies raised against platelet-derived growth factor (PDGF), the observed effects of platelet-released supernatants were diminished. The mitogenic response was also decreased when extracellular signal-regulated protein kinase (ERK) signalling was inhibited by PD98059; however, PD98059 did not reverse the effects of platelet-released supernatants on migration and osteogenic differentiation. Consistent with an ERK-mediated mitogenic activity, incubation of serum-starved mesenchymal cell progenitors with platelet-released supernatants increased phosphorylation of the kinase. Together, these observations indicate that PDGF is a key factor released upon platelet activation that can increase migration and proliferation, and decreases osteogenic differentiation of mesenchymal progenitor cells under in vitro conditions. The results further suggest that ERK signalling is required to mediate the mitogenic response to platelet-released supernatants.