Reinhard Gruber

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.

Platelets are mitogenic for periosteum-derived cells

The early stages of bone regeneration are associated with a high mitogenic activity of periosteal cells. Here we addressed the question of whether platelets that accumulate within the developing haematoma can account for this tissue response. Addition of platelets, platelet‐released supernatants, platelet membranes, and microparticles to bovine periosteum‐derived cells resulted in an increase in 3H‐thymidine incorporation; lipid extracts had no effect. Platelet‐released supernatants retained their activity after incubation at 56°C, but not at 100°C. Gel chromatographic analysis revealed the highest mitogenic activity at approximately 35 kD. Of the factors released from activated platelets, basic fibroblast growth factor (bFGF) and platelet‐derived growth factor (PDGF) increased 3H‐thymidine incorporation. The mitogenic activity of platelet‐released supernatants was decreased by anti‐PDGF, and anti‐bFGF antibodies. Platelet‐released supernatants increased the number of proliferating periosteum‐derived cells as determined by the expression pattern of Ki67. Platelet‐released supernatants also resulted in a stimulation of cell proliferation in periosteal explants. These results suggest that platelets have the potential to stimulate the mitogenic response of the periosteum during bone repair.

Expression of the vitamin D receptor, of estrogen and thyroid hormone receptor α- and β-isoforms, and of the androgen receptor in cultures of native mouse bone marrow and of stromal/osteoblastic cells

Marrow stromal cells mediate the effect of 1alpha,25-dihydroxyvitamin D3 on formation of osteoclast-like cells from undifferentiated hematopoetic precursors in bone marrow. Induction by the vitamin D hormone of multinucleated, calcitonin receptor- and tartrate-resistant acid phosphatase-positive cells in primary mouse bone marrow culture can be modulated by other members of the steroid/thyroid hormone family, such as triiodothyronine, which has a positive effect, as well as 17beta-estradiol and 5alpha-dihydrotestosterone, which both act as inhibitors of osteoclastogenesis. In an attempt to relate these effects of the steroid/thyroid hormones to the presence of their respective nuclear receptors, we studied expression of the vitamin D receptor (VDR), estrogen receptor (ER)-alpha and -beta, thyroid hormone receptor (TR)-alpha and -beta, and androgen receptor (AR) in total bone marrow as well as primary marrow stromal cell cultures. By using reverse-transcriptase-polymerase chain reaction, in both cases amplification products were obtained, which were identified by multiple restriction fragment length analysis as transcripts from mRNA specific for the ligand-binding domains of the VDR, ER-alpha, ER-beta, TR-alpha, TR-beta, and AR. Specific immunostaining by indirect peroxidase labeling revealed that among the various cell types present in bone marrow, the steroid/ thyroid hormone receptors are abundant particularly in marrow stromal cells. In another series of experiments, we extended our survey on receptor expression also to stromal/osteoblastic cell lines. At the mRNA level, the complete repertoire of steroid/thyroid hormone receptors was present in preadipocytic ST2 cells as well as in osteoblastic MC3T3-E1 cells. By immunocytochemical staining of the latter, it became apparent that single cells exhibit wide variations in intensity of specific signals for all the receptors investigated, so that, notably in contrast to primary stromal cells and ST2 cells, MC3T3-E1 display a mosaic pattern of receptor protein expression.

Platelet-released supernatants stimulate formation of osteoclast-like cells through a prostaglandin/RANKL-dependent mechanism

Platelets are activated at fracture sites or upon the insertion of implants as a consequence of vascular disruption and secrete the contents of their granules into the developing hematoma. The regeneration of injured tissue requires bone remodeling and the resorbing activity of osteoclasts. To test our hypothesis that platelets can stimulate osteoclastogenesis, we examined the effects of supernatants released from thrombin-activated platelets on osteoclast-like cell formation in murine bone marrow cultures. Histochemical analysis indicated the presence of bone-resorbing, tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Transcripts that are characteristically expressed in native osteoclasts were increased in these cultures, as determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. The inhibition of both cyclooxygenases with indomethacin, as well as the addition of the cyclooxygenase-2 (COX-2)-selective antagonist, NS398, completely blocked osteoclast-like cell formation and decreased endogenous prostaglandin E(2) production. Platelet-released supernatants stimulated the expression of receptor activator of NF-kappaB ligand (RANKL), whereas mRNA levels of osteoprotegerin (OPG) were decreased. The formation of osteoclast-like cells was prevented by recombinant OPG. Our results suggest that COX-2 activity is necessary for osteoclast-like cell formation in response to platelet-released supernatants, and that endogenously produced prostaglandin E(2) can, in turn, increase the RANKL:OPG ratio, indicating that platelets can contribute to bone remodeling by stimulation of osteoclastogenesis.

17Beta-estradiol antagonizes effects of 1alpha,25-dihydroxyvitamin D3 on interleukin-6 production and osteoclast-like cell formation in mouse bone marrow primary cultures.

In mouse bone marrow primary cultures, the formation of osteoclast-like, i.e. tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells (MNC), when induced by 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), can be suppressed by 17β-estradiol (17β-E2), whereas 17α-E2 is without any effect. 17β-E2, above 10−11 m, significantly reduced 1α,25(OH)2D3-mediated TRAP+ MNC formation in cultured bone marrow cells from both female and male mice. The estrogen at 10−8 m suppressed the peak response to the vitamin D sterol by 50%. 17β-E2 significantly suppressed basal and 1α,25(OH)2D3-stimulated cellular production of interleukin (IL)-6. IL-6 alone, although bone marrow cells in hormone-free culture produced appreciable amounts of the cytokine, did not induce any TRAP+ MNC. Therefore, the changes in IL-6 production induced by the hormones could not be the sole determinant for the extent of TRAP+ MNC formation. However, the stimulatory effect of 1α,25(OH)2D3 on osteoclastogenesis nevert...

Phenotypic Heterogeneity of Osteoblast-like MC3T3-E1 Cells: Changes of Bradykinin-Induced Prostaglandin E2 Production During Osteoblast Maturation

We have examined clonal murine calvarial MC3T3‐E1 cells obtained from different sources to compare their osteoblastic features (alkaline phosphatase [ALP], cyclic adenosine monophosphate [cAMP] response to parathyroid hormone, prostaglandin E2 (PGE2) and PGE1, bradykinin‐induced production of PGE2). It was found that the sublines investigated showed large variation of the above‐mentioned parameters, which may be attributed to distinct differentiated stages of osteoblast development. Increase of ALP activity was paralleled by an increase in cAMP accumulation in response to the above‐mentioned agents. The most striking difference was observed with bradykinin‐induced production of PGE2. Early stage cells (low ALP) produced high levels of PGE2, whereas cells with high ALP activity showed no bradykinin stimulation at all. This was consistent with the results of specific binding of3H‐bradykinin to its receptor and also correlated well with the bradykinin‐induced signal transduction sequence (inositol triphosphate liberation and elevation of intracellular calcium levels). This was confirmed by Northern blot analysis of bradykinin receptor mRNA expression. These results indicate that the widely used osteoblast‐like cell line MC3T3‐E1 is synonymous for multiple sublines, representing different stages of osteoblast development. These sublines were most likely emerging from the early stage cell line due to the applied culture conditions. Moreover, distinct biochemical features are displayed in correlation to the differentiation stage, thus providing a useful model to study the molecular mechanism of osteoblast maturation.

Interaction of triiodothyronine with 1α, 25-dihydroxyvitamin D3 on interleukin-6-dependent osteoclast-like cell formation in mouse bone marrow cell cultures

In mouse bone marrow cultures, the formation of osteoclast-like, that is, tartrate-resistant acid phosphatase-positive (TRAP+) and calcitonin (CT) receptor-positive multinucleated cells (MNCs), induced by 10(-10) to 10(-8) mol/L 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], could be augmented by triiodothyronine (T3), which alone had no effect on osteoclast-like cell formation. The permissive effect of T3 increased the response to 1alpha,25(OH)2D3 by approximately one order of magnitude. Linear concentration dependence was observed between 10(-11) and 10(-8) mol/L T3. Importantly, inhibition of prostaglandin synthesis by indomethacin significantly impeded osteoclast-like cell formation by 1alpha,25(OH)2D3 and abrogated the effect of T3 thereon. Basal interleukin-6 (IL-6) production by cultured marrow cells was significantly stimulated by 1alpha,25(OH)2D3. However, even at an exceedingly high concentration of 20 ng/mL, IL-6 was ineffective in inducing osteoclast-like cell formation. Therefore, any hormonally induced rise in IL-6 release from bone marrow cells could not account for the observed changes in TRAP+ MNC numbers. Nevertheless, the stimulatory effect of 1alpha,25(OH)2D3 on osteoclastogenesis was partially dependent on IL-6 because it could be significantly blocked by a neutralizing monoclonal anti-IL-6 antibody, and to the same extent by a monoclonal anti-IL-6 receptor antibody. Unimpaired signaling through the IL-6/IL-6R system is also a prerequisite for the auxiliary effect of T3 on induction of osteoclast-like cells by 1alpha,25(OH)2D3. Our data provide evidence that 1alpha,25(OH)2D3 induces osteoclast-like cell formation, at least in part, in an IL-6-dependent mode of action, which is also subject to modulation by T3. The mechanism of interaction of the two hormones apparently involves joint stimulation of prostaglandin synthesis.

BMP-6-induced osteogenic differentiation of mesenchymal cell lines is not modulated by sex steroids and resveratrol.

Bone morphogenetic protein-6 (BMP-6) is a potent inducer of osteogenic differentiation and its expression is stimulated by 17beta-estradiol. The existence of a regulatory loop between sex steroids and BMP-6 is therefore reasonable to hypothesize. Here we determined whether the sex steroids 17beta-estradiol and dihydrotestosterone, and the phytoestrogen resveratrol can modulate BMP-6-induced alkaline phosphatase activity and osteocalcin expression. Mesenchymal cells of murine (osteoblastic MC3T3-E1 cells, preadipogenic ST2 cells, prechondrogenic ATDC5 cell) and human origin (osteosarcoma SaOS and HOS cells, primary bone marrow stromal cells) were cultured in the presence of recombinant BMP-6 under serum-free conditions. BMP-6 dose-, and time-dependently increased alkaline phosphatase activity in murine cell lines, but not in human cells. Osteocalcin expression was also increased upon stimulation with BMP-6. The presence of 17beta-estradiol, dihydrotestosterone, and resveratrol had no effect on BMP-6-induced alkaline phosphatase activity and osteocalcin expression. These data suggest that osteogenic differentiation in response to BMP-6 occurs independent of steroid hormones and resveratrol in mesenchymal cells that express basal receptor levels.

Effects of cartilage-derived morphogenetic proteins and osteogenic protein-1 on osteochondrogenic differentiation of periosteum-derived cells

Localization studies and genetic evidence have implicated Cartilage-derived morphogenetic proteins-1, -2 (CDMP-1 and CDMP-2) and osteogenic protein-1 (OP-1) in the osteochondrogenic differentiation of mesenchymal progenitor cells during embryonic development and in postnatal life. Based on their expression pattern and the evidence that periosteum contains mesenchymal cells in the cambium layer that can undergo bone and cartilage formation, we hypothesized that CDMPs and OP-1 may be involved in long bone development and fracture healing. To test this hypothesis, periosteum-derived cells from young calves were cultured as monolayers under serum-free conditions with and without the addition of recombinant CDMP-1, CDMP-2 and OP-1. Phenotypic analysis indicate that periosteum-derived cell populations prepared, expanded and cultured under the conditions described below, constitutively express mRNAs for the bone markers osteocalcin, osteopontin and collagen type I, and the chondrogenic markers collagen type II and aggrecan as determined by reverse transcription (RT)-PCR. Moreover, histologic examinations showed positive staining for alcian blue and alkaline phosphatase (AP). Treatment of periosteum-derived cells with CDMPs and OP-1 resulted in a dose-dependent increase of cell proliferation; CDMP-2 was less active in this regard. Furthermore, all growth factors enhanced osteogenic differentiation as assessed by a time- and dose-dependent stimulation of AP activity and OP-1 increased mRNA expression for osteocalcin and collagen type I. We further examined the effects of CDMPs and OP-1 on chondrogenic differentiation of periosteum-derived cells. Both CDMPs and OP-1 stimulated 35S-sulfate incorporation into newly synthesized macromolecules with OP-1 having a more pronounced stimulatory effect when compared with CDMP-1 and CDMP-2. Our results indicate that distinct members of the BMP-family act on periosteum-derived cells to increase their mitotic and metabolic activity. The enhancement of both the chondrogenic and osteogenic differentiation suggests that these growth factors might contribute to the postnatal local regulation of bone formation and fracture repair.

Bisphosphonat-assoziierte Osteonekrosen des Kieferknochens

SummaryIntravenous application of bisphosphonates (BP) represents an established therapeutic strategy of tumor-associated bone metastasis and severe hypercalcemia. Patients can develop osteonecrosis of the jaw (ONJ) as a side effect of this therapy. The diagnosis of ONJ is based on three criteria: a) patients have been or are currently treated with BP; b) a deficiency in wound healing, which is in 70–80% associated with necrotic alveolar bone, characteristically exposed, is present for at least 8 weeks and c) no radiotherapy of the head and neck was performed. The suppression of bone turnover, concomitant with high functional load of the alveolar bone, and the subsequent accumulation of microfractures are considered the main pathologic factors of this disease. The cumulative incidence of ONJ lies approximately between 1 and 10% in oncologic patients, being associated with the antiresorptive potency and the respective molecular structure of the BPs. Patients with multiple myeloma develop ONJ more frequently than patients with other oncological diseases such as metastasizing breast- and prostate cancer, a fact that may also be due to the higher transfusion/injection frequency of BP in these patients. Dental treatment strategies are responsible for the occurrence of ONJ in approximately 80% of cases. Based on a clinical staging, patients can be grouped into three categories and should receive the corresponding treatment regime. Prospective clinical studies are required for a better understanding of etiology and pathogenesis of ONJ to make treatment, risk estimation and prognosis of ONJ more accurate.ZusammenfassungDie intravenöse Verabreichung von Bisphosphonaten (BP) ist eine etablierte Basistherapie bei der Behandlung von Tumor-assoziierten Knochenmetastasen (osteoklastischen und osteoplastischen Metastasen) sowie schwerer Hyperkalzämien. Als unerwartete Komplikation dieser Behandlung wurde das Auftreten von Osteonekrosen im Kieferknochen (engl. BP-associated osteonecrosis of the jaw; ONJ) beschrieben. Die Diagnose der ONJ basiert auf drei Kriterien: a) die Patienten müssen mit BP behandelt worden sein; b) eine Wundheilungsstörung in der Mundhöhle, in 70–80% assoziiert mit einem exponierten, nekrotischen Kieferknochen, besteht länger als 8 Wochen und c) eine vorangegangene Radiotherapie des Kopf/Halsbereiches hat nicht stattgefunden. Die Pathogenese der ONJ steht im möglichen Zusammenhang mit der Akkumulation von Mikrofrakturen, die als Folge eines eingeschränkten Knochenumbaus, bei gleichzeitig hoher funktioneller Belastung des Kieferknochens, auftreten. Die kumulative Inzidenz der ONJ beträgt etwa 1 bis 10% bei Tumorpatienten, wobei ein Zusammenhang mit der antiresorptiven Potenz der einzelnen BP sowie deren Molekülstruktur besteht. Schon nach 2–3 Infusionen/Injektionen kann eine ONJ auftreten. Diese Komplikation tritt bei Patienten mit Multiplem Myelom häufiger auf als bei Patienten mit anderen onkologischen Grunderkrankungen, wie metastasiertem Mamma- oder Prostatakarzinom, was allerdings auch mit dem häufigeren Einsatz von BP bei Multiplem Myelom verbunden werden kann. Zahnärztliche Eingriffe sind in 80% auslösend für die Entstehung der ONJ. Patienten werden im Sinne eines klinischen Stagings in drei Kategorien eingeteilt und ein entsprechendes Behandlungskonzept angewandt. Weiterführende prospektive klinische Studien sind erforderlich, um die Ätiopathogenese besser zu verstehen und darauf aufbauend die Behandlungsstrategien zu optimieren und Risikofaktoren und Prognose besser abschätzen zu können.Intravenous application of bisphosphonates (BP) represents an established therapeutic strategy of tumor-associated bone metastasis and severe hypercalcemia. Patients can develop osteonecrosis of the jaw (ONJ) as a side effect of this therapy. The diagnosis of ONJ is based on three criteria: a) patients have been or are currently treated with BP; b) a deficiency in wound healing, which is in 70-80% associated with necrotic alveolar bone, characteristically exposed, is present for at least 8 weeks and c) no radiotherapy of the head and neck was performed. The suppression of bone turnover, concomitant with high functional load of the alveolar bone, and the subsequent accumulation of microfractures are considered the main pathologic factors of this disease. The cumulative incidence of ONJ lies approximately between 1 and 10% in oncologic patients, being associated with the antiresorptive potency and the respective molecular structure of the BPs. Patients with multiple myeloma develop ONJ more frequently than patients with other oncological diseases such as metastasizing breast- and prostate cancer, a fact that may also be due to the higher transfusion/injection frequency of BP in these patients. Dental treatment strategies are responsible for the occurrence of ONJ in approximately 80% of cases. Based on a clinical staging, patients can be grouped into three categories and should receive the corresponding treatment regime. Prospective clinical studies are required for a better understanding of etiology and pathogenesis of ONJ to make treatment, risk estimation and prognosis of ONJ more accurate.