Platelet Lysate Induces in Human Osteoblasts Resumption of Cell Proliferation and Activation of Pathways Relevant for Revascularization and Regeneration of Damaged Bone.

To understand the regenerative effect of platelet-released molecules in bone repair one should investigate the cascade of events involving the resident osteoblast population during the reconstructive process. Here the in vitro response of human osteoblasts to a platelet lysate (PL) stimulus is reported. Quiescent or very slow dividing osteoblasts showed a burst of proliferation after PL stimulation and returned to a none or very slow dividing condition when the PL was removed. PL stimulated osteoblasts maintained a differentiation capability in vitro and in vivo when tested in absence of PL. Since angiogenesis plays a crucial role in the bone healing process, we investigated in PL stimulated osteoblasts the activation of hypoxia-inducible factor 1-alpha (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) pathways, involved in both angiogenesis and bone regeneration. We observed phosphorylation of STAT3 and a strong induction, nuclear translocation and DNA binding of HIF-1α. In agreement with the induction of HIF-1α an enhanced secretion of vascular endothelial growth factor (VEGF) occurred. The double effect of the PL on quiescent osteoblasts, i.e., resumption of proliferation and activation of pathways promoting both angiogenesis and bone formation, provides a rationale to the application of PL as therapeutic agent in post-traumatic bone repair.

LCN2 overexpression in bone enhances the hematopoietic compartment via modulation of the bone marrow microenvironment.

Lipocalin-2 (LCN2) is a member of the lipocalin family whose expression is modulated in several conditions, including cell differentiation, innate immunity, stress, and cancer. Although it is known that it is expressed in bone, its function in this tissue remains poorly studied. To this end, we took advantage of transgenic mice lines that expressed LCN2 driven by a bone specific type I collagen (LCN2-Tg). In the bone marrow (BM) of LCN2-Tg mice we observed an increased number of phenotypically long-term hematopoietic stem cells (LT-HSC) that also displayed a higher proliferation rate compared to wild-type controls (Wt). Furthermore, hematopoietic progenitor cells, obtained from LCN2-Tg BM showed an increased clonogenic capacity compared to those obtained from LCN2-Tg spleen, a higher concentration of serum erythropoietin and a higher number of mature erythrocytes in the peripheral blood of old LCN2-Tg animals compared to aged-matched wt. The findings of a combined increase in the BM of the LCN2-Tg mice of SDF-1, SCF, and TIMP-1 levels along with the reduction of both MMP-9 activity and cathepsin K concentration may explain the observed effects on the HSC compartment. This study shows that LCN2 overexpression in bones modifies the BM microenvironment via modulation of the expression of key secreted factors and cytokines, which in turn regulate the HSC niche behavior enhancing both HSC homing in young mice and erythrocytes production in older mice.

Lipocalin-2 controls the expression of SDF-1 and the number of responsive cells in bone.

Lipocalin-2 (LCN2) is a member of the lipocalin family, small secreted proteins functioning as modulators of many different physiological processes including cell differentiation, proliferation and apoptosis. LCN2 expression is also up-regulated in several pathological conditions, including inflammation and cancer. LCN2 synthesis has been described in epithelia, bone and cells of the immune system. Despite its wide expression the role of LCN2 remains to be fully elucidated. To better understand the role of this lipocalin in the bone/bone marrow system we generated transgenic mice over-expressing LCN2 specifically in bone under the control of a type I collagen promoter. In the bone marrow of these transgenic mice we observed an increased expression of SDF-1 that correlated with an increased number of CD34+/CXCR4+ (SDF-1 receptor) cells. To some extent, this appeared due to an enhanced cell proliferation rate. The higher level of the factor synthesis and the increased number of cells expressing its receptor was maintained during animal aging. Our results show that LCN2 could play a role in determining the number of CD34+/CXCR4+ precursor cells in the bone marrow thus contributing to the control of the bone marrow microenvironment.

Platelet Lysate Inhibits NF-κB Activation and Induces Proliferation and an Alert State in Quiescent Human Umbilical Vein Endothelial Cells Retaining Their Differentiation Capability.

: Injured blood vessel repair and blood circulation re-establishment are crucial events for tissue repair. We investigated in primary cultures of human umbilical vein endothelial cells (HUVEC), the effects of platelet lysate (PL), a cocktail of factors released by activated platelets following blood vessel disruption and involved in the wound-healing process triggering. PL exerted a protective effect on HUVEC in an inflammatory milieu by inhibiting IL-1α-activated NF-κB pathway and by inducing the secretion of PGE2, a pro-resolving molecule in the wound microenvironment. Moreover, PL enhanced HUVEC proliferation, without affecting their capability of forming tube-like structures on matrigel, and activated resting quiescent cells to re-enter cell cycle. In agreement with these findings, proliferation-related pathways Akt and ERK1/2 were activated. The expression of the cell-cycle activator Cyclin D1 was also enhanced, as well as the expression of the High Mobility Group Box-1 (HMGB1), a protein of the alarmin group involved in tissue homeostasis, repair, and remodeling. These in vitro data suggest a possible in vivo contribution of PL to new vessel formation after a wound by activation of cells resident in vessel walls. Our biochemical study provides a rationale for the clinical use of PL in the treatment of wound healing-related pathologies.

p38/NF-kB-dependent expression of COX-2 during differentiation and inflammatory response of chondrocytes.

Studying cartilage differentiation, we observed the emergence of inflammation-related proteins suggesting that a common pathway was activated in cartilage differentiation and inflammation. In the present paper, we investigated the expression pathway of the inflammation-related enzyme Cyclooxygenase-2 (COX-2) during differentiation and inflammatory response of the chondrocytic cell line MC615. Cells were cultured either as (i) proliferating prechondrogenic cells expressing type I collagen or (ii) differentiated hyperconfluent cells expressing Sox9 and type II collagen. The p38 and the NF-kB pathways were investigated in standard conditions and after inflammatory agents treatment. NF-kB was constitutively activated in differentiated cells. The activation level of NF-kB in differentiated cells was comparable to the level in proliferating cells treated with the inflammatory agent LPS. In both cases, p65 was bound to the NF-kB consensus sequence of COX-2 promoter. p38, constitutively activated in differentiated cells, was activated in proliferating cells by treatment with LPS or IL-1alpha. In stimulated proliferating cells the two pathways are connected since addition of the p38-specific inhibitor SB203580 inhibited p38 activation, significantly reduced NF-kB activation and repressed COX-2 synthesis indicating that p38 is upstream NF-kB activation and COX-2 synthesis. In differentiated cells, the treatment with the inflammatory agent neither enhance NF-kB activation, nor synthesis of COX-2 while the addition of SB203580 neither repressed activation of p38, nor COX-2 synthesis, suggesting a constitutive activation of a p38/NF-kB/COX2 pathway. Our data indicate that in chondrocytes, COX-2 is expressed via p38 activation/NF-kB recruitment during both differentiation and inflammatory response.

Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1.

The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage.

Platelet Lysate Activates Human Subcutaneous Adipose Tissue Cells by Promoting Cell Proliferation and Their Paracrine Activity Toward Epidermal Keratinocytes.

Skin chronic wounds are non-healing ulcerative defects, which arise in association with a morbidity state, such as diabetes and vascular insufficiency or as the consequence of systemic factors including advanced age. Platelet Rich Plasma, a platelet-rich blood fraction, can significantly improve the healing of human skin chronic ulcers. Given that the subcutaneous adipose tissue is located beneath the skin and plays a role in the skin homeostasis, in this study, we investigated the in vitro response of human subcutaneous adipose tissue cells to platelet content in a model mimicking in vitro the in situ milieu of a deep skin injury. Considering that, at the wound site, plasma turn to serum, platelets are activated and inflammation occurs, human adipose-derived stromal cells (hASC) were cultured with Human Serum (HS) supplemented or not with Platelet Lysate (PL) and/or IL-1α. We observed that HS sustained hASC proliferation more efficiently than FBS and induced a spontaneous adipogenic differentiation in the cells. PL added to HS enhanced hASC proliferation, regardless the presence of IL-1α. In the presence of PL, hASC progressively lessened the adipogenic phenotype, possibly because the proliferation of less committed cells was induced. However, these cells resumed adipogenesis in permissive conditions. Accordingly, PL induced in quiescent cells activation of the proliferation-related pathways ERK, Akt, and STAT-3 and expression of Cyclin D1. Moreover, PL induced an early and transient increase of the pro-inflammatory response triggered by IL-1α, by inducing COX-2 expression and secretion of a large amount of PGE2, IL-6, and IL-8. Media conditioned by PL-stimulated hASC exerted a chemotactic activity on human keratinocytes and favored the healing of an in vitro scratch wound. In order to bridge the gap between in vitro results and possible in vivo events, the stimuli were also tested in ex vivo cultures of in toto human adipose tissue biopsies (hAT). PL induced cell proliferation in hAT and outgrowth of committed progenitor cells able to differentiate in permissive conditions. In conclusion, we report that the adipose tissue responds to the wound microenvironment by activating the proliferation of adipose tissue progenitor cells and promoting the release of factors favoring wound healing.

Learning from Mother Nature: Innovative Tools to Boost Endogenous Repair of Critical or Difficult-to-Heal Large Tissue Defects.

For repair of chronic or difficult-to-heal tissue lesions and defects, major constraints exist to a broad application of cell therapy and tissue engineering approaches, i.e., transplantation of "ex vivo" expanded autologous stem/progenitor cells, alone or associated with carrier biomaterials. To enable a large number of patients to benefit, new strategies should be considered. One of the main goals of contemporary regenerative medicine is to develop new regenerative therapies, inspired from Mother Nature. In all injured tissues, when platelets are activated by tissue contact, their released factors promote innate immune cell migration to the wound site. Platelet-derived factors and factors secreted by migrating immune cells create an inflammatory microenvironment, in turn, causing the activation of angiogenesis and vasculogenesis processes. Eventually, repair or regeneration of the injured tissue occurs via paracrine signals activating, mobilizing or recruiting to the wound site cells with healing potential, such as stem cells, progenitors, or undifferentiated cells derived from the reprogramming of tissue differentiated cells. This review, largely based on our studies, discusses the identification of new tools, inspired by cellular and molecular mechanisms overseeing physiological tissue healing, that could reactivate dormant endogenous regeneration mechanisms lost during evolution and ontogenesis.

Mesenchymal stem cell paracrine activity is modulated by platelet lysate: induction of an inflammatory response and secretion of factors maintaining macrophages in a proinflammatory phenotype.

Wound healing is achieved through distinct programmed phases: hemostasis, inflammation, mesenchymal cell proliferation and migration, and tissue remodeling. At the injury site, clot formation and platelet degranulation release cytokines and growth factors and actively participating in the healing process and regulating the migration of inflammatory cells, such as neutrophils, macrophages, and lymphocytes. We previously demonstrated that, in an inflammatory environment, prostaglandin E2 (PGE2) secreted by mesenchymal stem cells (MSCs) promoted the macrophage switch from a proinflammatory to a proresolving phenotype. Using an in vitro model, we here evaluated the role carried out by the two main players of the wound healing process, the platelet degranulation content mimicked by the platelet lysate (PL) and the inflammatory stimulus, on the modulation of mouse bone-marrow-derived MSC paracrine activity. We demonstrated that, in MSCs, PL induced nuclear factor kappaB (NF-κB) activation, expression of COX-2 and mPGE synthase, and PGE2 production; in an inflammatory microenvironment, PL increased the inflammatory response and promoted the secretion of the proinflammatory cytokine IL-6. We assayed on mouse primary macrophages the paracrine activity of MSCs exposed to the different microenvironments and we observed that PL-treated MSC-conditioned medium maintained macrophages in a proinflammatory state. The involved factors were granulocyte macrophage-colony stimulating factor induced by PL in MSCs and TNF-α induced by PL-MSC-conditioned medium in macrophages. Our findings indicate that PL triggers an inflammatory response in MSCs and induces the secretion of factors maintaining macrophages in a proinflammatory state thus enhancing the initial inflammatory response to the injury, a key element in the activation of wound healing.

The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair.

In vivo implanted bone marrow-derived mesenchymal stem cells trigger a cascade of cellular events leading to the formation of an ectopic bone regenerative niche.

We recently reported that mouse bone marrow stromal cells, also known as bone marrow (BM)-derived mesenchymal stem cells (MSCs), seeded onto a scaffold and implanted in vivo, led to an ectopic bone deposition by host cells. This MSCs capacity was critically dependent on their commitment level, being present only in MSCs cultured in presence of fibroblast growth factor-2. Taking advantage of a chimeric mouse model, in this study we show that seeded MSCs trigger a cascade of events resulting in the mobilization of macrophages, the induction of their functional switch from a proinflammatory to a proresolving phenotype, and the subsequent formation of a bone regenerative niche through the recruitment, within the first 2 weeks of implantation, of endothelial progenitors and of cells with an osteogenic potential (CD146+CD105+), both of them derived from the BM. Moreover, we demonstrated that, in an inflammatory environment, MSCs secrete a large amount of prostaglandin E2 playing a key role in the macrophage phenotype switch.

Platelet-rich plasma exerts antinociceptive activity by a peripheral endocannabinoid-related mechanism.

In regenerative medicine, platelet by-products containing factors physiologically involved in wound healing, have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects. Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide, 2-arachidonoylglycerol, palmitoylethanolamide, and oleoylethanolamide. Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that, under inflammatory conditions, PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue. Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated a potent antinociceptive effect against both early and late nocifensive responses. This effect was observed following intrapaw injection of (1) total PRP; (2) lipids extracted from PRP; and (3) an endocannabinoid-enriched lipid fraction of PRP. In all conditions, antagonists of endocannabinoid CB1 and CB2 receptors, injected in the paw, abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action. In conclusion, we showed that PRP and PRP lipid extract exert a potent antinociceptive activity linked, at least in part, to their endocannabinoids and related compound content, and to their capability of elevating the levels of these lipid mediators in cells.

Platelet lysate induces in vitro wound healing of human keratinocytes associated with a strong proinflammatory response.

Platelet lysates (PL), which are derived from platelets, are cocktails of growth factors and cytokines that can promote tissue regeneration. Until today, most studies have focused on growth factor content of platelets rather than on their potential as a reservoir of mediators and cytokines. Taking advantage of an in vitro scratch assay performed under both normal and inflammatory conditions, in the present work, we report that at physiologic concentrations, PL enhanced wound closure rates of NCTC 2544 human keratinocytes. This effect was clearly detectable 6 h after wounding. Moreover, PL induced a strong cell actin cytoskeletal re-organization that persisted up to 24 h. The accelerated wound closure promoted by PL, in either presence or absence of serum, was associated with a high expression of the inflammatory cytokine interleukin-8. Further, after 24 h PL treatment, confluent keratinocytes also expressed low amounts of interleukin-8 and of the antimicrobial peptide neutrophil gelatinase-associated lipocalin, which dramatically increased under inflammatory conditions. These effects were associated with activation of the inflammatory pathways, p38 mitogen-activated protein kinase, and NF-κB. Our findings support the concept that platelet-derived preparations could accelerate regeneration of difficult-to-heal wounds by triggering an inflammatory cascade and having an antimicrobial role.

Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities.

Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in vitro under iron-limiting conditions. The 1.8 Å crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding copurified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities.