Locally delivered rhTGF-beta2 enhances bone ingrowth and bone regeneration at local and remote sites of skeletal injury.

The purposes of the present study were to determine if recombinant human transforming growth factor-beta-2 (rhTGF-beta2) enhances bone ingrowth into porous-coated implants and bone regeneration in gaps between the implant and surrounding host bone. The implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs in the presence of a 3-mm gap. In three treatment groups of animals, the test implant was treated with hydroxyapatite/tricalcium phosphate (HA/TCP) and rhTGF-beta2 in buffer at a dose per implant of 1.2 microg (n = 6), 12 microg (n = 7), or 120 microg (n = 7) and placed in the left humerus. In these same animals, an internal control implant treated only with HA/TCP and buffer was placed in the right humerus. In a non-TGF-beta treated external control group of animals (n = 7), one implant was treated with HA/TCP while the contralateral implant was not treated with the ceramic. In vitro analyses showed that approximately 15%, of the applied dose was released within 120 h with most of the release occurring in the first 24 h. The TGF-beta treated implants had significantly more bone ingrowth than the controls with the greatest effect in the 12 microg/implant group (a 2.2-fold increase over the paired internal control (P = 0.004) and a 4-fold increase over the external control (P < 0.001)). The TGF-beta treated implants had significantly more bone formation in the gap than the controls with the greatest effect in the 12 and 120 microg groups (1.8-fold increases over the paired internal controls (P = 0.003 and P = 0.012, respectively) and 2.8-fold increases over the external controls (P < 0.001 and P = 0.001, respectively)). Compared to the external controls, the internal control implants tended to have more bone ingrowth (1.9-fold increase, P = 0.066) and had significantly more bone formation in the gap (1.7-fold increase. P = 0.008). Thus, application of rhTGF-beta2 to a porous-coated implant-stimulated local bone ingrowth and gap healing in a weakly dose-dependent manner and stimulated bone regeneration in the 3-mm gap surrounding the contralateral control implant, a site remote from the local treatment with the growth factor.

Recombinant human interleukin-11 directly promotes megakaryocytopoiesis in vitro.

We have investigated the mechanism of action of the thrombopoietic cytokine, recombinant human interleukin-11 (rhIL-11), on megakaryocytopoiesis in vitro. We have shown that rhIL-11-induced murine and human megakaryocytopoiesis are not mediated by thrombopoietin (Tpo). Murine megakaryocytes (MKs) were produced from bone marrow (BM) mononuclear cells cultured with rhIL-11, IL-3, and a combination of the two cytokines. Conditioned media (CM) were collected and assayed for the presence of biologically active Tpo. Tpo activity was not detected in any of the CMs tested. Next, human BM CD34+ cells were cultured in serum-free fibrin clot medium with rhIL-11, IL-3, or rhIL-11 plus IL-3 and an antibody that neutralizes human Tpo activity. No inhibition of either burst-forming unit-MK- or colony-forming unit-MK-derived colony formation was observed. The antibody did partially inhibit steel factor-induced MK-colony formation, suggesting that the actions of this cytokine are mediated, in part, by Tpo. We determined that MKs can be direct targets of rhIL-11 by showing the expression of functional IL-11 receptor on these cells. Total RNA was prepared from cultured human BM CD41+CD14- cells (MKs) and IL-11 receptor alpha chain mRNA was detected in the MKs by reverse transcription-polymerase chain reaction. Analysis of single-sorted CD41+CD14- cells confirmed that the observed IL-11 receptor expression was not due to contaminating CD41- cells in the pool. The presence of rhIL-11 receptor alpha chain protein in the cells was established by Western blot analysis. After a short exposure of purified BM MKs to rhIL-11, enhanced phosphorylation of both its signal transduction subunit, gp130, and the transcription factor, STAT3 was detected, showing a direct activation of receptor signaling by the cytokine. Consistent with the lack of effect of rhIL-11 on platelets in vivo, IL-11 receptor alpha chain mRNA and protein were not detected in isolated human platelets. These data indicate that rhIL-11 acts directly on MKs and MK progenitors but not on platelets.

Effect of recombinant interleukin-6 and thrombopoietin on isolated guinea pig bone marrow megakaryocyte protein phosphorylation and proplatelet formation.

Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100ng/mal IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 microg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAK1 and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation.

Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading.

Guinea pig bone marrow megakaryocytes were cultured on a type I rat tail collagen gel which stimulated proplatelet formation. Proplatelet formation was inhibited by monoclonal antibody LM609 to the alpha v beta 3 integrin (VnR), but not by monoclonal antibodies to the alpha 5, alpha 6, beta 1, or IIb beta 3(GPIIb-IIIa) integrin proteins. Megakaryocytes cultured on a plastic surface and stimulated with thrombin undergo a spreading and an adhesion reaction. This reaction is blocked in a dose-dependent manner by the tetrapeptide RGDS and by the monoclonal antibody PG2 to the GPIIb-IIIa integrin, but not by the monoclonal antibody LM609 to the VnR. Immunoprecipitation and affinity chromatography experiments demonstrate that guinea pig megakaryocytes have distinct GPIIb-IIIa and VnR integrins with similar electrophoretic mobility. Spreading was significantly inhibited in a dose-dependent fashion by drugs which elevate cellular cyclic AMP, including forskolin, dibutyryl cAMP, and isobutylmethylxanthine. In contrast to spreading, megakaryocyte proplatelet formation was stimulated by these agents in a dose-dependent manner. Megakaryocyte spreading was stimulated by the protein kinase C (PKC) activator phorbol myristate acetate (PMA) and inhibited by the PKC inhibitors Calphostin C and K5720 in a dose-dependent manner. PKC inhibitors did not inhibit megakaryocyte proplatelet formation. These results demonstrate that the closely related VnR and GPIIb-IIIa integrins regulate different aspects of megakaryocyte morphological change and appear to be associated with different second messenger systems.

Megakaryocyte proplatelet-like process formation in vitro is inhibited by serum prothrombin, a process which is blocked by matrix-bound glycosaminoglycans.

The process of platelet shedding from megakaryocytes is incompletely understood, due in part to the impossibility of studying this dynamic process in vivo. Megakaryocytes in situ and in in vitro cultures display extended cytoplasmic processes constricted at platelet-sized intervals which presumably are the structural intermediates between megakaryocytes and platelets. This study describes the establishment of a serum-free culture system of purified guinea pig megakaryocytes in which extensive cytoplasmic process formation can be observed on 21 to 29% of the cells. The addition of as little as 0.05% pooled human serum to the cultures will completely but reversibly block process development. The serum inhibitor was identified as residual prothrombin, which upon contact with megakaryocytes is converted to the serine esterase thrombin. Thrombin directly prevents the formation of new processes and also induces retraction of existing processes. When megakaryocytes are cultured on Matrigel, process formation occurs even in an excess of thrombin. This potentiation of process development in the presence of inhibitory factors is mediated by the glycosaminoglycan content of Matrigel. The physiological implications of these observations are discussed.

Extracellular matrix stimulation of guinea pig megakaryocyte proplatelet formation in vitro is mediated through the vitronectin receptor.

We have used an in vitro culture system to study the role of extracellular matrix (ECM) in the fragmentation of guinea pig bone marrow megakaryocytes (MK) and the formation of proplatelet fragments from these cells. Proplatelet formation is stimulated by culturing the cells on a hydrated type I collagen gel in the presence of serum. MK express integrin proteins alpha 5, alpha 6, beta 1, and the alpha v beta 3 complex as demonstrated by immunofluorescence. A monoclonal antibody, LM 609, to the alpha v beta 3 vitronectin receptor blocked proplatelet formation, whereas the monoclonal antibodies to the beta 1, alpha 5, and alpha 6 integrin proteins did not inhibit proplatelet formation. The tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits proplatelet formation; however, there was no inhibition by the fibronectin receptor-specific peptide GRGDdSP. The fibrinogen gamma chain peptide HHLGGAKQAGDV, which binds to the platelet membrane glycoprotein complex IIb-IIIa but not to the vitronectin receptor (VnR), did not inhibit proplatelet formation, nor did two different laminin peptides. In the absence of serum, 5.7% of MK spontaneously formed processes or fragmented. The addition of 50 micrograms/ml of vitronectin to serum-free cultures increased proplatelet formation to 21.5% of the MK, equal to cultures with 10% serum. Stimulation of proplatelet formation by vitronectin in serum-free cultures was inhibited by LM 609. Antibody staining with anti-bovine vitronectin antibody showed that MK contain intracellular vitronectin. These data show that guinea pig MK express alpha 5, alpha 6, beta 1, and alpha v beta 3 integrins. However, only the MK vitronectin receptor and its interaction with vitronectin plays an essential role in proplatelet formation in vitro.

Lipid and membrane fluidity abnormalities in platelets and megakaryocytes of the hereditary macrothrombocytopenic Wistar Furth rat.

Biochemical and functional abnormalities of megakaryocytes and platelets were studied in Wistar Furth (WF) rats which have genetically determined macrothrombocytopenia and megakaryocytopenia, and were compared with their counterparts in Sprague-Dawley (SD) rats. Both megakaryocytes and platelets synthesized phospholipids from [14C]acetate. WF and SD megakaryocytes incorporated 0.27 and 0.29 nmol acetate per 10(6) cells, respectively. Phosphatidylcholine (PC) accounted for 64% and 58% of the PL radioactive label in megakaryocytes of SD and WF rats, respectively, (P less than 0.05), while 69% of labeled activity was associated with PC of SD platelets compared to 60% found in PC of WF platelets (P less than 0.01). In WF platelets a significant increase in the levels of lysophosphatidylcholine (6.1% vs. 3.0%) was observed. WF platelets had substantially higher levels of esterified cholesterol, triglycerides, ceramides and a 3-fold increase in the total protein per platelet compared to SD platelets. The fatty acid composition of WF platelet PC showed quantitative abnormalities. Plasma lecithin-cholesterol acyl transferase activity and platelet function monitored by the uptake and release of [14C] serotonin showed nonsignificant variations between SD and WF rats. Compared with the control, platelet membrane fluidity, measured by fluorescence polarization using platelets labeled with 1,6-diphenyl-1,3,5-hexatriene, was significantly decreased in the WF rats.

Immunomagnetic bead isolation of megakaryocytes from guinea-pig bone marrow: effect of recombinant interleukin-6 on size, ploidy and cytoplasmic fragmentation.

Guinea-pig bone marrow megakaryocytes were isolated using an antibody to platelet glycoprotein Ib and a second antibody conjugated to magnetic beads. The procedure yielded an average of 644,800 megakaryocytes from two guinea-pigs with an average viability of 83%. All of the platelet glycoprotein Ib positive cells also expressed the platelet glycoprotein IIb-IIIa complex. The size and ploidy of megakaryocytes isolated by this technique were analysed in the presence of 10 ng/ml of interleukin-6 (IL-6). Without IL-6 megakaryocyte size increased significantly after 24 h, but an even larger increase in size occurred in the presence of IL-6. The modal ploidy class was 16N with an average of 19% 2N, 2.6% 4N, 16.4% 8N, 50.8% 16N and 11.1% 32N cells as determined by flow cytometry. Measurements made by microspectrophotometry were in close agreement. After 24 h incubation there was a significant rise in the percentage of 2N and 32N cells. The ploidy distribution after 24 h with IL-6 was the same as the control. Megakaryocytes cultured in the absence of serum on collagen gels did not form pseudopods and fragment, as occurs with serum (Leven et al, 1987). Addition of IL-6 to the serum-free cultures caused megakaryocytes to form extensive proplatelet extensions. We conclude that large numbers of pure guinea-pig bone marrow megakaryocytes can be isolated by immunomagnetic bead selection, including low ploidy immature megakaryocytes. Spontaneous maturation occurred as evidenced by the increase in megakaryocyte size and ploidy. IL-6 altered megakaryocyte size and morphology but not ploidy, indicating that these different characteristics of megakaryocytes may be regulated separately.

Blood platelet formation in vitro. The role of the cytoskeleton in megakaryocyte fragmentation.

We have developed a unique in vitro model that promotes differentiation of megakaryocytes into platelets. When megakaryocytes isolated from guinea pig bone marrow were cultured on hydrated rat tail collagen gels, cells spontaneously formed elongated, beaded processes that fragmented to yield cytoplasmic pieces with the same size and internal composition as individual platelets. Addition of nocodazole at the initiation of cultures blocked process formation, while addition of nocodazole to cells with previously established processes resulted in their retraction. The addition of taxol to cultures resulted in abnormally thick processes that were tightly adherent to the underlying substratum, and did not bead or fragment. Cytochalasin D accelerated process formation and fragmentation of megakaryocytes cultured on collagen gels by twofold. On the basis of these results, we propose a model for platelet formation in culture that involves the following steps: adherence of megakaryocytes to the underlying extracellular matrix; dilation of the demarcation membrane system and breakdown of the actin-rich peripheral zone; microtubule-based extension of pseudopodia, which are no longer adherent to the substratum; and fragmentation into platelets by the coalescence and fusion of demarcation membrane vesicles with the plasma membrane. We feel that this distinctive culture system closely approximates thrombocytopoiesis in vivo, thus allowing detailed elucidation of this important process.

Collagenase production by guinea pig megakaryocytes in vitro.

Cultured guinea pig bone marrow megakaryocytes were found to secrete a 92-kd collagenase that was detected by digestion of gelatin in a polyacrylamide substrate gel assay. Neither casein or bovine serum albumin were digested by this enzyme. The enzyme is a neutral metalloprotease. Its secretion is increased by thrombin (1.0 U/ml) and phorbol myristate acetate (10 ng/ml) and is unaffected by prostaglandin E1 (10 microM). In the absence of serum, gelatinase secretion is inhibited, but it can be stimulated by cytochalasin D (1.0 microgram/ml). Gelatinase activity in the medium from megakaryocytes cultured on rat tail collagen gel is decreased. Medium from megakaryocytes cultured on Matrigel contains a second gelatinase of 90 kd. Addition of the tetrapeptide RGDS to the cultures on Matrigel blocks the appearance of the 90-kd gelatinase. Platelets contained both a 92- and a 90-kd gelatinase that was detected only after thrombin activation. The results indicate that megakaryocytes can secrete a collagenase, and its secretion may be in part controlled by interaction with the extracellular matrix. The appearance of the 90-kd gelatinase may be associated with megakaryocyte maturation and platelet formation.

The effect of partially purified thrombopoietin on guinea pig megakaryocyte ploidy in vitro.

Enriched populations of guinea pig bone marrow megakaryocytes were prepared by density gradient and velocity centrifugation and maintained in liquid cultures for 24 or 48 h. The resulting megakaryocyte preparations were of 86.1% +/- 5.5% purity. After 24 or 48 h in liquid culture, recovery of viable cells was 77% +/- 11% or 83% +/- 13%, respectively. Megakaryocyte cultures were supplemented with 100-200 micrograms/ml of either a lectin-fractionated preparation of thrombopoietin (TPO) from the plasma of thrombocytopenic rabbits or an identically prepared protein fraction from non-thrombocytopenic animals. Addition of TPO resulted in a significant increase (p less than 0.05) in both the proportion and total numbers of 32N megakaryocytes and a significant decrease (p less than 0.05) in the relative frequency of 8N megakaryocytes. In most experiments, a decrease in the total number of 8N megakaryocytes also was noted. These results indicate that partially purified TPO is able to increase the ploidy (DNA levels) of megakaryocytes in vitro.

Megakaryocyte and platelet ultrastructure in the Wistar Furth rat.

Wistar Furth (WF) rats were studied and compared with Sprague-Dawley (SD) rats to determine if ultrastructural abnormalities in platelets or megakaryocytes could explain their macrothrombocytopenia. WF rats had one third of the platelet count of healthy rats and two times the platelet volume. Megakaryocyte number was decreased and the size of mature stage three megakaryocytes also was decreased. WF platelets had large membranous inclusions, and otherwise showed normal ultrastructural morphology. The WF megakaryocytes showed abnormal aggregates of the demarcation membrane system. Ruthenium red staining was more intense on WF megakaryocytes and platelets, indicating a possible increase in surface mucopolysaccharides. It is possible that abnormal megakaryocyte membrane structure may lead to WF macrothrombocytopenia.

Megakaryocyte motility and platelet formation.

The mechanism of platelet formation is reviewed with special emphasis on the role of the cytoskeleton. The three major theories for platelet formation are by cytoplasmic budding, cytoplasmic dissolution or pseudopod formation. Most evidence indicates that platelets form as fragments of megakaryocyte pseudopodia. Pseudopodia formation is stimulated in vitro by thrombocytopenic rabbit plasma. It is inhibited by vincristine and altered by taxol. Cytochalasins cause pseudopodia to form in isolated megakaryocytes. Therefore, normal pseudopodia formation may depend on a combination of microfilament disorganization and microtubule elongation.

Megakaryocyte morphogenesis stimulated in vitro by whole and partially fractionated thrombocytopenic plasma: a model system for the study of platelet formation.

Isolated guinea pig megakaryocytes were cultured in the presence of plasma from normal or thrombocytopenic rabbits. Thrombocytopenic but not normal plasma stimulated formation of long cytoplasmic processes and cytoplasmic fragmentation. Activity was found in the 60% to 80% ammonium sulfate fraction of thrombocytopenic plasma but not in the 0% to 60% fraction. The 60% to 80% fraction of normal plasma contained a small amount of activity. Both colchicine and vincristine inhibited the morphogenesis stimulated by thrombocytopenic plasma. Cytochalasin B and D both mimicked the thrombocytopenic plasma-induced morphological change and affected more megakaryocytes than did the thrombocytopenic plasma. Cytochalasin and thrombocytopenic plasma together had a synergistic effect, causing many megakaryocytes to form processes and break into cytoplasmic fragments 3 to 6 microns in diameter. Immunofluorescence staining with antitubulin antiserum showed that cytoplasmic processes formed in the presence of thrombocytopenic plasma contain microtubules and that fragments released by the megakaryocytes contain microtubule rings. A model for the cytoskeletal basis of platelet formation is proposed.

Fibrinogen biosynthesis in isolated guinea pig megakaryocytes.

Fibrinogen synthesis was investigated in guinea pig megakaryocytes. Purified megakaryocytes were incubated with 35S-methionine in methionine-free incubation medium for 18 hours. Newly synthesized fibrinogen in megakaryocyte lysates enriched with purified carrier guinea pig fibrinogen was immunoprecipitated with a specific anti-guinea pig fibrinogen antiserum produced in rabbits. Proteins in the immunoprecipitates were analyzed with a 3.5% to 10.0% gradient polyacrylamide slab gel electrophoresis and auto-radiography. Radioactivity was detected in a protein band of 340,000 daltons. In order to verify fibrinogen synthesis, immunoprecipitate was analyzed by two-dimensional slab gel electrophoresis: (1) the first dimension separated unreduced fibrinogen using a 3.5% to 10.0% gradient gel; (2) following reduction by 2-beta-mercaptoethanol, fibrinogen chains were separated in the second dimension using a 10% gel. Alpha, beta, and gamma fibrinogen chains, which represented carrier guinea pig plasma fibrinogen, were visualized by Coomassie brilliant blue. Autoradiography identified the incorporation of radioactivity into the three fibrinogen chains. In control experiments, immunoprecipitates, produced by exposing megakaryocyte lysates to preimmune rabbit serum and goat anti-rabbit IgG, were also analyzed by the two-dimensional gel system. Radioactivity was not detected in sites corresponding to the migration of fibrinogen subunits. The study demonstrates that isolated guinea pig megakaryocytes can synthesize fibrinogen. The electrophoretic mobility of newly synthesized fibrinogen and subunits is similar to that of guinea pig plasma fibrinogen.

Alpha-actinin arcs in megakaryocyte spreading.

Cultured megakaryocytes, isolated from guinea pig bone marrow, were treated with buffer or adenosine diphosphate (ADP) (10 microM) on plain or coated glass surfaces. Control cells were rounded and non-adherent. The nucleotide induced the cells to spread to several times the initial diameter, and to become flattened and markedly adherent to the substratum. 'Cytoskeletons' were examined by transmission electron microscopy (TEM). Those from unspread cells contained only rare microfilaments and no filament bundles; those from spread cells contained large numbers of microfilaments in nets and many filament bundles, which were largely oriented circumferentially. Interference reflection microscopy demonstrated that the spread cells were attached to the substratum in arc-shaped regions, which corresponded to arcs containing alpha-actinin as seen by specific immunofluorescence of the same cells. However, other arcs of alpha-actinin staining did not correspond to arcs of tight attachment. We conclude that fibrous arcs, which appear to assemble as part of the spreading process, play a role in what are probably transient surface attachment sites. A survey of observations of spreading in other cell types suggests that similar arcs are more widespread than has been realized.

Platelet shape change and cytoskeletal assembly: effects of pH and monovalent cation ionophores.

The monovalent cation ionophores monensin and nigericin cause platelet shape change at a rate of approximately 1/20 of that caused by ADP. The effect of monensin was studied further. Shape change caused by monensin is pH dependent, increasing in rate as extracellular pH increases. Monensin induced shape change is not blocked by 30 microM cinanserin which completely inhibits serotonin induced shape change. Also, the amount of serotonin secreted by monensin treated platelets is below the threshold required to induce shape change. 100 microM ATP which inhibits ADP induced shape change does not affect monensin induced shape change. Amiloride, a sodium transport blocker, inhibits both the rate of ADP induced shape change and platelet spreading on poly-lysine coated glass. Amorphous platelet cytoskeletons isolated from resting platelets at pH 6.8 with Mg++ but not Ca++ can be transformed into filament bundles by subsequent incubation at pH 7.6. We conclude that platelet shape change is at least in part triggered by changes in cellular Na+ and pH.

Role of sodium in ADP- and thrombin-induced megakaryocyte spreading.

We investigated the role of sodium in megakaryocyte spreading induced by thrombin and ADP. We found that if extracellular sodium was replaced by lithium, potassium, or choline, spreading was inhibited. When extracellular sodium was present, amiloride or tetrodotoxin inhibited spreading. Using intracellular recording we found spreading to be associated with a permanent membrane depolarization. The extent and rate of thrombin-induced depolarization was reduced when lithium replaced sodium. Unspread cells had an average membrane potential of -44.8 mV. Spread cells had an average membrane potential of -18.46 mV. When choline replaced sodium, or when in the presence of tetrodotoxin and amiloride, the spread cells repolarized, indicating that the depolarization is due to an increase in sodium permeability. Similar treatments did not change the membrane potential of unspread cells. Incubation of megakaryocytes with A23187 together with monensin or methylamine induced spreading. Methylamine occasionally caused spreading by itself, but neither ionophore alone caused spreading. These results indicate that megakaryocyte spreading induced by ADP and thrombin depends on an increase in sodium conductance.