Early Stages Of Bone Regeneration Research Articles

Chirality-biased protein expression profile during early stages of bone regeneration.

Introduction: Chirality is a crucial mechanical cue within the extracellular matrix during tissue repair and regeneration. Despite its key roles in cell behavior and regeneration efficacy, our understanding of chirality-biased protein profile in vivo remains unclear. Methods: In this study, we characterized the proteomic profile of proteins extracted from bone defect areas implanted with left-handed and right-handed scaffold matrices during the early healing stage. We identified differentially-expressed proteins between the two groups and detected heterogenic characteristic signatures on day 3 and day 7 time points. Results: Proteomic analysis showed that left-handed chirality could upregulate cell adhesion-related and GTPase-related proteins on day 3 and day 7. Besides, interaction analysis and in vitro verification results indicated that the left-handed chiral scaffold material activated Rho GTPase and Akt1, ultimately leading to M2 polarization of macrophages. Discussion: In summary, our study thus improved understanding of the regenerative processes facilitated by chiral materials by characterizing the protein atlas in the context of bone defect repair and exploring the underlying molecular mechanisms of chirality-mediated polarization differences in macrophages.

Molecular Basis beyond Interrelated Bone Resorption/Regeneration in Periodontal Diseases: A Concise Review.

Periodontitis is the sixth most common chronic inflammatory disease, destroying the tissues supporting the teeth. There are three distinct stages in periodontitis: infection, inflammation, and tissue destruction, where each stage has its own characteristics and hence its line of treatment. Illuminating the underlying mechanisms of alveolar bone loss is vital in the treatment of periodontitis to allow for subsequent reconstruction of the periodontium. Bone cells, including osteoclasts, osteoblasts, and bone marrow stromal cells, classically were thought to control bone destruction in periodontitis. Lately, osteocytes were found to assist in inflammation-related bone remodeling besides being able to initiate physiological bone remodeling. Furthermore, mesenchymal stem cells (MSCs) either transplanted or homed exhibit highly immunosuppressive properties, such as preventing monocytes/hematopoietic precursor differentiation and downregulating excessive release of inflammatory cytokines. In the early stages of bone regeneration, an acute inflammatory response is critical for the recruitment of MSCs, controlling their migration, and their differentiation. Later during bone remodeling, the interaction and balance between proinflammatory and anti-inflammatory cytokines could regulate MSC properties, resulting in either bone formation or bone resorption. This narrative review elaborates on the important interactions between inflammatory stimuli during periodontal diseases, bone cells, MSCs, and subsequent bone regeneration or bone resorption. Understanding these concepts will open up new possibilities for promoting bone regeneration and hindering bone loss caused by periodontal diseases.

One-Step Preparation of an Injectable Hydrogel Scaffold System Capable of Sequential Dual-Growth Factor Release to Maximize Bone Regeneration.

Numerous growth factors are involved in the natural bone healing process, which is precisely controlled in a time- and concentration-dependent manner. Mimicking the secretion pattern of growth factors could be an effective means to maximize the bone regeneration effect. However, achieving the sequential delivery of various growth factors without the use of multiple materials or complex scaffold designs is challenging. Herein, an injectable poly(organophosphazene) hydrogel scaffold (IPS) encapsulating bone morphogenetic protein (BMP)-2 and TGFβ-1 (IPS_BT) is studied to mimic the sequential secretion of growth factors involved in natural bone healing. The IPS_BT system is designed to release TGFβ-1 slowly while retaining BMP-2 for a longer period of time. When IPS_BT is injected in vivo, the hydrogel is replaced by bone tissue. In addition, angiogenic (CD31and alpha-smooth muscle actin (α-SMA)) and stemness (Nanog andSOX2) markers are highly upregulated in the early stages of bone regeneration. The IPS system developed here has promising applications in tissue engineering because 1) various amounts of the growth factors can be loaded in one step, 2) the release pattern of each growth factor can be controlled via differences in their molecular interactions, and 3) the injected IPS can be degraded and replaced with regenerated bone tissue.

Multiple Ion Scaffold-Based Delivery Platform for Potential Application in Early Stages of Bone Regeneration.

Bone has the intrinsic capacity to regenerate itself, as long as the damage is small, through the sequential stimulation of specific phases, such as angiogenesis followed by osteogenesis. However, when the damage is extensive it is unable to regenerate and bone tissue engineering is used as an alternative. In this study, we developed a platform to allow the triple ion delivery with sequential delivery capacity to potentially stimulate antibacterial, angiogenic and osteogenic processes. The scaffold-based platform consisted of alginate/hydroxyapatite (HA) microparticles embedded in alginate fibers. Firstly, microparticles were developed using different ratios of alginate:HA using the spraying method, resulting in a high reproducibility of the technique. Microparticle size between 100–300 µm and ratio 1:40 resulted in a more spherical morphology and were selected for their incorporation into alginate fiber. Different amounts of copper and cobalt were added with the microparticles and alginate fiber, respectively, were used as model ions which could eventually modulate and mimic antimicrobial and angiogenic processes. Moreover, calcium ion was also incorporated in both, in order to provide the system with potential osteogenic properties together with HA. The multiple delivery of copper, cobalt and calcium released were in the therapeutic range as measured by induced coupled plasma (ICP), providing a promising delivery strategy for tissue engineering.

Notch-Wnt signal crosstalk regulates proliferation and differentiation of osteoprogenitor cells during intramembranous bone healing

Adult bone regeneration is orchestrated by the precise actions of osteoprogenitor cells (OPCs). However, the mechanisms by which OPC proliferation and differentiation are linked and thereby regulated are yet to be defined. Here, we present evidence that during intramembranous bone formation OPC proliferation is controlled by Notch signaling, while differentiation is initiated by activation of canonical Wnt signaling. The temporospatial separation of Notch and Wnt signal activation during the early stages of bone regeneration suggests crosstalk between the two pathways. In vitro and in vivo manipulation of the two essential pathways demonstrate that Wnt activation leads to initiation of osteogenic differentiation and at the same time inhibits Notch signaling, which results in termination of the proliferative phase. Here, we establish canonical Wnt signaling as a key regulator that facilitates the crosstalk between OPC proliferation and differentiation during intramembranous, primary bone healing.

Carbon nanohorn coating by electrodeposition accelerate bone formation on titanium implant

Direct contact between bone and implant materials is required for dental implants. Titanium is used for the implant material owing to its mechanical and biological properties. The anodisation as the surface treatment was employed to enhance osteogenesis around titanium. Moreover, carbon nanohorn (CNH), a type of nanometer-sized carbon material, was reported to promote the bone formation. Thus, it is expected that if the surface of anodised Ti (AnTi) is modified with CNHs, Ti-bone contact would be enhanced. In this study, the Ti surface was modified with CNHs by electrophoresis and obtained anodised titanium coated with CNHs (CNH/AnTi). In vitro, CNH/AnTi attracted osteoblastic cells more than AnTi, thereby the proliferation of osteoblastic cell was enhanced by CNH/AnTi more than by AnTi. In vivo, at 7 and 28 days after implantation of CNH/AnTi or AnTi into the rat femur, more aggressive bone formation was observed on the surface of CNH/AnTi than on AnTi. More importantly, the area where newly formed bone tissue directly attached to CNH/AnTi was significantly larger than that for AnTi, suggesting that “contact osteogenesis” was accelerated on CNH/AnTi during the early post-implantation period. CNH/AnTi would be advantageous especially for the early stages of bone regeneration after surgery.

Synthesis and in vitro Evaluation of Thermosensitive PLA-g-P(HEM-co-NIPAAM) Hydrogel Used for Delivery of VEGF

Bone has self-healing potential, but this characteristic is limited and requires external intervention. Bone formation is a dynamic process influenced by various growth factors. Angiogenesis is a fundamental phase and essential in the early stages of bone regeneration. Because of insufficient vascularization within osteoconductive or osteoinductive bone scaffolds, VEGF can be loaded into the scaffolds structure to induce blood vessels throughout engineered tissue. For this propose, PLA-g-P(HEM-co-NIPAAM) copolymers with HEMA:NIPAAm ratio of 1:1 and 1:5 are synthesized, and their biocompatibility, swelling, and vascular endothelial growth factor release properties are investigated. The hydrogels were biocompatible, and the cell attachment and growth were increased facing these hydrogels. The swelling performance of the synthesized hydrogels is increased by increasing the PHEMA ratio in the copolymer, which leads to high loading capacity. Different specifications for percent released over different time periods were achieved for the as-synthesized hydrogel, which will play a powerful role in bone regeneration in the near future.

OZONE IMPROVES AUTOGENOUS GRAFT HEALING IN EXPERIMENTAL DIABETES MELLITUS: A MORPHOMETRIC AND IMMUNOHISTOCHEMICAL STUDY

Backround: Diabetes mellitus (DM) is a metabolic disorder which plays crucial role in the pathogenesis of periodontitis. Ozone have several actions such as antimicrobial ,immunostimulating, antihypoxic effects, and activates antioxidant enzymes and angiogenesis Aim : The aim of this study is to investigate the effect of gaseous ozone on bone healing in diabetic rat calvarial defects treated with autogenous bone graft, morphometrically and immunohistochemically. Material and Methods: Diabetes was induced and critical size defects were created on rats. Study groups: 1-Empty defect (Control, n=14) group, 2-Autograft (AG, n=14) group, 3-Empty defect+ozone therapy (Control+Ozone, n=14) group, 4-Autograft+ozone application (AG+Ozone, n=14) group. Gaseous ozone was applied on the operation day and the following 2 weeks daily ( 140ppm @ 2L/d, 2.24 mg ). Total bone area was measured. Osteocalcin and Bone morphogenic protein-2 protein expressions were evaluated . Results : Control and Control+Ozone groups had no osteoclast and residuel lacunae during the study . Osteoblasts in AG+Ozone group were higher than AG group at 4th week (p>0.05). AG+Ozone group had more total bone area than AG group at 4th week. AG+Ozone group revealed more BMP-2 immune positivity compared to the other groups. Osteocalcin immune positivity in AG groups was higher than those of the Control groups. Conclusion : Within the limitations of this study, gaseous ozone application decreased osteoclast number and increased osteoblast number and bone regeneration, especially, in early stages of bone regeneration in diabetic rats. KEYWORDS: autograft, bone regeneration , calvarial defect , experimental diabetes mellitus , ozone

Allogeneic Chondrogenic Mesenchymal Stromal Cells Alter Helper T Cell Subsets in CD4+ Memory T Cells.

Implantation of chondrogenically differentiated mesenchymal stromal cells (MSCs) leads to bone formation in vivo through the process of endochondral ossification. The use of allogeneic MSCs for this purpose may be a promising new approach to replace the current gold standard of bone regeneration. However, the success of using allogeneic cells depends on the interaction between the implanted cells and the host's endogenous immune cells. Th17 T cells and other CD4 helper T cell subtypes have been shown to negatively impact chondrogenesis, however, it is unclear how the interaction between these cells affects bone regeneration mediated by these cells. The aim of the current work was to assess the effect of chondrogenic MSC pellets on Th1, Th2, Th17, and regulatory T cells in vitro. Human MSCs were nonchondrogenic (-TGFβ3) and chondrogenically (+TGFβ3) differentiated for 7 or 21 days. Memory T cells (sorted from the CD4 population of peripheral blood mononuclear cells [PBMCs]), as well as total PBMCs were cocultured with allogeneic nonchondrogenic and chondrogenic MSC pellets for 3 days. Seven-day differentiated allogeneic nonchondrogenic and chondrogenic MSC pellets that were cocultured with memory T cells resulted in a significant increase in Th2 and a decrease in Th1 T cells. Furthermore, the co-culture of 21-day differentiated nonchondrogenic and chondrogenic MSC pellets with memory T cells resulted in a significant increase in Th2 and Th17 T cells, as well as a decrease in Th1 and regulatory T cells. Interleukin (IL)-6 was identified as a predominant cytokine involved in this interaction between allogeneic chondrogenically differentiated MSC pellets and memory CD4 T cells, with high levels of IL-6 being secreted in the supernatants of this cocultured condition. The findings of this study highlight the potential of chondrogenically differentiated MSC pellets to alter the ratio of Th1 and Th2 as well as Th17 and regulatory T cell subsets. Additional analysis investigating bone formation by chondrogenically differentiated MSCs in an allogeneic setting may identify a novel role of these T cell subsets in bone regeneration processes mediated by chondrogenically differentiated MSCs. Impact statement Allogeneic mesenchymal stromal cells (MSCs) have the potential to be an off-the-shelf treatment for bone repair. However, the lack of knowledge of the immune cells involved in this process has hampered the progression to the clinic. The current study has shown that allogeneic chondrogenic MSCs have the potential to skew the ratio of specific helper CD4 T cell subsets in vitro. This has now provided insight for future in vivo experiments to investigate the role of these T cell subsets in the early stages of bone regeneration mediated by allogeneic chondrogenic MSCs.

Cell behavior under hypoxic conditions. Computational 3D model

During the early stages of bone regeneration, oxygen plays a key role, recruiting mesenchymal stem cells and regulating the processes of differentiation, proliferation, and apoptosis. To study in these effects, a 3D computational model has been developed, where the effects of oxygen in the mentioned processes are considered.

CaP Coating and Low-Level Laser Therapy to Stimulate Early Bone Formation and Improve Fixation of Rough Threaded Implants.

This study aimed to compare in vivo osteogenesis on rough threaded dental implants with and without calcium phosphate (CaP) coating deposition, alone or in association with low-level laser therapy (LLLT) by gallium aluminum arsenide. Four groups were studied: G1: implant; G2: implant + CaP coating; G3: implant + LLLT; and G4: implant + CaP coating + LLLT. LLLT was applied for 7 days at the surgical site before and after placing the implant. Topographic characterization was performed before surgery using scanning electron microscopy and energy dispersion spectrophotometry. Bone-implant contact (BIC) was measured after 1, 2, and 6 weeks and reverse torque after 6 weeks. In short periods, G2, G3, and G4 showed significantly greater BIC than G1 (P < 0.05), but no difference in BIC was observed at 6 weeks. However, the values for the removal torque test at 6 weeks were higher in G2 and G4 (P < 0.05). Both CaP coating alone and using LLLT induce cellular stimulation and improve BIC in short-term healing, resulting in higher implant fixation, and should be considered in clinical practice due to their low cost and high effectiveness.

Delayed bone healing by collagen membrane in early phase of 4 weeks

Barrier membranes are important in maintaining space in guided bone regeneration process by preventing downgrowth of epithelial or connective tissue. In this study, the effects of resorbable membranes during the early stages of bone regeneration in rats with impaired bone healing capacity were investigated. Twenty-eight rats were selected for this study. Half of the animals were selected for radiation therapy before surgical procedure (G3, G4). Animals were assigned into 4 groups (G1-G4). A circular defect was created in the central parietal bone. It was covered with resorbable membrane in G2 and G4. After 4 weeks, the animals were sacrificed. At week 4, the new bone formation was observed around the margin of old bone in G1, G2 and G4 groups. Osteoclast was most abundant in the G1 group (18.3 ± 7.7) and least abundant in the G4 group (7.9 ± 4.7). The mean of osteocalcin levels in blood was the highest in the G2 group and lowest in the G3 group. Only G4 group showed significant difference in Runx2 levels between before-treatment and after- treatment. Bone healing is adversely affected after radiation therapy. In addition, resorbable membranes can delay healing in the early stages of bone regeneration.

Protein expression during early stages of bone regeneration under hydrophobic and hydrophilic titanium domes. A pilot study.

There is significant evidence that, during the early stages of osseointegration, moderately rough hydrophilic (SLActive) surfaces can accelerate osteogenesis and increase bone-to-implant contact in comparison to hydrophobic (SLA) surfaces. However, very little is known regarding the molecular mechanisms behind the influence that surface chemistry modifications to increase hydrophilicity determine on bone healing. The aim of this study was to describe for the first time the proteins and related signalling pathways expressed during early osseous healing stages under SLA and SLActive titanium domes for guided bone regeneration. One SLA and 1 SLActive dome with an internal diameter of 5.0mm and a height of 3.0mm were secured to the parietal bones of nine 6-month-old male New Zealand rabbits. Three animals were randomly euthanized at 4, 7 and 14days and the newly formed tissues retrieved under the domes were analysed with liquid chromatography-mass spectrometry/mass spectrometry. STRING and KEGG databases were applied for Gene Ontology and pathway analyses. A different modulation of several pathways was detected between the 2 groups at all healing times. The main differences in the osseous healing response associated to the 2 surfaces were related to pathways involved in regulating the inflammatory response, differentiation of osteoblast precursors and skeletogenesis. At day 7, the highest number of proteins and the highest cellular activity were observed in both groups, although a more complex and articulated proteome in terms of cellular metabolism and signal transduction was observed in SLActive samples. This is the first study describing the proteome expressed during early healing stages of guided bone regeneration and osseointegration. A combination of enhanced early osteogenic response and reduced inflammatory response were suggested for the hydrophilic group. Future studies are needed to corroborate these findings and explore the molecular effects of different titanium surfaces on the cascade of events taking place during bone formation.

Modeling early stage bone regeneration with biomimetic electrospun fibrinogen nanofibers and adipose-derived mesenchymal stem cells

AbstractThe key events of the earliest stages of bone regeneration have been described in vivo although not yet modeled in an in vitro environment, where mechanistic cell-matrix-growth factor interactions can be more effectively studied. Here, we explore an early-stage bone regeneration model where the ability of electrospun fibrinogen (Fg) nanofibers to regulate osteoblastogenesis between distinct mesenchymal stem cells populations is assessed. Electrospun scaffolds of Fg, polydioxanone (PDO), and a Fg:PDO blend were seeded with adipose-derived mesenchymal stem cells (ASCs) and grown for 7-21 days in osteogenic differentiation media or control growth media. Scaffolds were analyzed weekly for histologic and molecular evidence of osteoblastogenesis. In response to osteogenic differentiation media, ASCs seeded on the Fg scaffolds exhibit elevated expression of multiple genes associated with osteoblastogenesis. Histologic stains and scanning electron microscopy demonstrate widespread mineralization within the scaffolds, as well as de novo type I collagen synthesis. Our data demonstrates that electrospun Fg nanofibers support ASC osteogenic differentiation, yet the scaffold itself does not appear to be osteoinductive. Together, ASCs and Fg recapitulate early stages of bone regeneration ex vivo and presents a prospective autologous therapeutic approach for bone repair.

Evaluation of chemically modified SLA implants (modSLA) biofunctionalized with integrin (RGD)‐ and heparin (KRSR)‐binding peptides

Enhancing osseointegration through surface immobilization of multiple short peptide sequences that mimic extracellular matrix (ECM) proteins, such as arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), has not yet been extensively explored. Additionally, the effect of biofunctionalizing chemically modified sandblasted and acid-etched surfaces (modSLA) is unknown. The present study evaluated modSLA implant surfaces modified with RGD and KRSR for potentially enhanced effects on bone apposition and interfacial shear strength during early stages of bone regeneration. Two sets of experimental implants were placed in the maxillae of eight miniature pigs, known for their rapid wound healing kinetics: bone chamber implants creating two circular bone defects for histomorphometric analysis on one side and standard thread configuration implants for removal torque testing on the other side. Three different biofunctionalized modSLA surfaces using poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) as a carrier minimizing nonspecific protein adsorption [(i) 20 pmol cm⁻² KRSR alone (KRSR); or in combination with RGD in two different concentrations; (ii) 0.05 pmol cm⁻² RGD (KRSR/RGD-1); (iii) 1.26 pmol cm⁻² RGD (KRSR/RGD-2)] were compared with (iv) control modSLA. Animals were sacrificed at 2 weeks. Removal torque values (701.48-780.28 N mm), bone-to-implant contact (BIC) (35.22%-41.49%), and new bone fill (28.58%-30.62%) demonstrated no significant differences among treatments. It may be concluded that biofunctionalizing modSLA surfaces with KRSR and RGD derivatives of PLL-g-PEG polymer does not increase BIC, bone fill, or interfacial shear strength.

Bone morphogenetic protein 2 stimulates endochondral ossification by regulating periosteal cell fate during bone repair

Bone repair depends on the coordinated action of numerous growth factors and cytokines to stimulate new skeletal tissue formation. Among all the growth factors involved in bone repair, Bone Morphogenetic Proteins (BMPs) are the only molecules now used therapeutically to enhance healing. Although BMPs are known as strong bone inducers, their role in initiating skeletal repair is not entirely elucidated. The aim of this study was to define the role of BMP2 during the early stages of bone regeneration and more specifically in regulating the fate of skeletal progenitors. During healing of non-stabilized fractures via endochondral ossification, exogenous BMP2 increased the deposition and resorption of cartilage and bone, which was correlated with a stimulation of osteoclastogenesis but not angiogenesis in the early phase of repair. During healing of stabilized fractures, which normally occurs via intramembranous ossification, exogenous BMP2 induced cartilage formation suggesting a role in regulating cell fate decisions. Specifically, the periosteum was found to be a target of exogenous BMP2 as shown by activation of the BMP pathway in this tissue. Using cell lineage analyses, we further show that BMP2 can direct cell differentiation towards the chondrogenic lineage within the periosteum but not the endosteum, indicating that skeletal progenitors within periosteum and endosteum respond differently to BMP signals. In conclusion, BMP2 plays an important role in the early stages of repair by recruiting local sources of skeletal progenitors within periosteum and endosteum and by determining their differentiation towards the chondrogenic and osteogenic lineages.

Removal Torque Values of Titanium Implants Inserted into Bone Defects Filled with Hydroxyapatite: A Histologic and Histomorphometric Analysis in Rabbit

The successful clinical use of implants with microrough titanium surfaces has paved the way for the further development of surface topographies to promote enhanced peri-implant bone apposition during the early stages of bone regeneration. The aim of this study is to determine the torque resistance of the implant-bone interface of plasma sprayed titanium endosseous implants placed in the rabbit tibia in three different situations. Hydroxyapatite (HA) particles with a size ranging from 250 to 450 microm were used in the present study to fill out 6 mm bony defects created in rabbit tibia. Fourteen mature New Zealand white male rabbits, weighing about 2.5 Kg., were used in this study. The test sites were filled with HA and the non-grafted sites were used as control. A total of 28 bone defects (14 without HA and 14 with HA.) were created. Implants with plasma-spray surface were subsequently inserted. Six additional rabbits were used; in these animals no bone defects were created. The implant sites were divided into three groups: 1) implants inserted in the tibia without bone defects; 2) implants inserted in the tibia with bone defects not filled with HA; 3) implants inserted in the tibia with bone defects filled with HA particles. The purpose of this study is to measure reverse-torque in three different situations of clinically similar implant geometry in the articulation of rabbits. The null hypothesis stated that there is a difference in reversal torque between the bone regeneration with HA, native bone and bone defect without bone regeneration. Additionally, comparison between these 6-month data and the 3-month data from a previous study led to the following conclusions: a significant difference exists in the removed torque for the TPS implants in the bone regenerated by HA and native bone; increase in removal torque values was seen for the implant inserted in bone regenerated by HA compared to the implant nated in bone without biomaterials. In conclusion, bone regenerated with biomaterials possesses different mechanical characteristics to native bone.

Enhanced bone apposition around biofunctionalized sandblasted and acid‐etched titanium implant surfaces

Microrough titanium (Ti) surfaces of dental implants have demonstrated more rapid and greater bone apposition when compared with machined Ti surfaces. However, further enhancement of osteoblastic activity and bone apposition by bio-functionalizing the implant surface with a monomolecular adsorbed layer of a co-polymer - i.e., poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its derivatives (PLL-g-PEG/PEG-peptide) - has never been investigated. The aim of the present study was to examine early bone apposition to a modified sandblasted and acid-etched (SLA) surface coated with an Arg-Gly-Asp (RGD)-peptide-modified polymer (PLL-g-PEG/PEG-RGD) in the maxillae of miniature pigs, and to compare it with the standard SLA surface. Test and control implants had the same microrough topography (SLA), but differed in their surface chemistry (polymer coatings). The following surfaces were examined histomorphometrically: (i) control - SLA without coating; (ii) (PLL-g-PEG); (iii) (PLL-g-PEG/PEG-RDG) (RDG, Arg-Asp-Gly); and (iv) (PLL-g-PEG/PEG-RGD). At 2 weeks, RGD-coated implants demonstrated significantly higher percentages of bone-to-implant contact as compared with controls (61.68% vs. 43.62%; P < 0.001). It can be concluded that the (PLL-g-PEG/PEG-RGD) coatings may promote enhanced bone apposition during the early stages of bone regeneration.

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 (H2O2) 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 H2O2. 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 H2O2. The capacity of platelets to release H2O2-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 H2O2, a process that may play a role during the early stages of bone regeneration.

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.