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Objective: Pigs are emerging as a preferred experimental in vivo model for bone regeneration. The study objective was to answer the focused PEO question: in the pig model (P), what is the capacity of experimental alveolar bone defects (E) for spontaneous regeneration in terms of new bone formation (O)? Methods: Following PRISMA guidelines, electronic databases were searched for studies reporting experimental bone defects or extraction socket healing in the maxillae or mandibles of pigs. The main inclusion criteria were the presence of a control group of untreated defects/sockets and the assessment of regeneration via 3D tomography [radiographic defect fill (RDF)] or 2D histomorphometry [new bone formation (NBF)]. Random effects meta‐analyses were performed for the outcomes RDF and NBF. Results: Overall, 45 studies were included reporting on alveolar bone defects or extraction sockets, most frequently in the mandibles of minipigs. Based on morphology, defects were broadly classified as 'box‐defects' (BD) or 'cylinder‐defects' (CD) with a wide range of healing times (10 days to 52 weeks). Meta‐analyses revealed pooled estimates (with 95% confidence intervals) of 50% RDF (36.87%–63.15%) and 43.74% NBF (30.47%–57%) in BD, and 44% RDF (16.48%–71.61%) and 39.67% NBF (31.53%–47.81%) in CD, which were similar to estimates of socket‐healing [48.74% RDF (40.35%–57.13%) and 38.73% NBF (28.57%–48.89%)]. Heterogeneity in the meta‐analysis was high (I2 > 90%). Conclusion: A substantial body of literature revealed a high capacity for spontaneous regeneration in experimental alveolar bone defects of (mini)pigs, which should be considered in future studies of bone regeneration in this animal model. [ABSTRACT FROM AUTHOR]

Bio-Gide® is a collagen membrane routinely used in guided bone regeneration. Recent studies have shown that this collagen membrane has osteoconductive properties, meaning that it can support the growth of new bone. However, it has also been observed that the collagen membrane has areas of mineralized fibers which can occur spontaneously and independently of osteoblasts. To better understand how this works, we established a model using minced collagen membranes to reduce the active mineralization of intact collagen membranes in favor of passive mineralization. We thus compared the original intact membrane with a minced collagen membrane in a 5 mm calvarial defect model in Sprague Dawley rats. After three weeks of healing, histology and microcomputed tomography (μCT) were performed. Histological analysis confirmed the osteoconductive properties, with new bone growing inside the intact collagen membrane. However, in minced collagen membranes, the osteoconductive properties were restricted to the defect margins. Interestingly, histology revealed large mineralized areas indicating passive mineralization with no signs of bone formation. In the μCT analysis, the intact collagen membranes caused a higher median mineralized volume (1.5 mm3) compared with the minced group (0.4 mm3), but this lacked significance (p = 0.09). The μCT analysis needs to be interpreted carefully, particularly in defects filled with minced membranes, considering that the mineralized tissue may not necessarily be bone but also the result of passive mineralization. Taken together, the findings suggest that Bio-Gide® collagen membranes support bone formation while also exhibiting potential for passive mineralization. [ABSTRACT FROM AUTHOR]

Based on the evidence of a significant communication and connection pathway between the bone and immune systems, a new science has emerged: osteoimmunology. Indeed, the immune system has a considerable impact on bone health and diseases, as well as on bone formation during grafts and its stability over time. Chronic inflammation induces the excessive production of oxidants. An imbalance between the levels of oxidants and antioxidants is called oxidative stress. This physio-pathological state causes both molecular and cellular damage, which leads to DNA alterations, genetic mutations and cell apoptosis, and thus, impaired immunity followed by delayed or compromised wound healing. Oxidative stress levels experienced by the body affect bone regeneration and maintenance around teeth and dental implants. As the immune system and bone remodeling are interconnected, bone loss is a consequence of immune dysregulation. Therefore, oral tissue deficiencies such as periodontitis and peri-implantitis should be regarded as immune diseases. Bone management strategies should include both biological and surgical solutions. These protocols tend to improve immunity through antioxidant production to enhance bone formation and prevent bone loss. This narrative review aims to highlight the relationship between inflammation, oxidation, immunity and bone health in the oral cavity. It intends to help clinicians to detect high-risk situations in oral surgery and to propose biological and clinical solutions that will enhance patients' immune responses and surgical treatment outcomes. [ABSTRACT FROM AUTHOR]

Objectives: Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth‐factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone‐marrow MSC (MSC‐CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte‐ and platelet‐rich fibrin (PRF‐CM), a current growth‐factor therapy, for guided bone regeneration (GBR). Methods: MSC‐CM and PRF‐CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC‐CM or PRF‐CM were applied on critical‐size rat calvaria defects and new bone formation was assessed via three‐dimensional (3D) micro‐CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks). Results: While both MSC‐CM and PRF‐CM revealed several bone‐related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC‐CM. In rat calvaria defects, micro‐CT revealed greater total bone coverage in the MSC‐CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC‐CM group, while the total new bone area was similar between groups. Conclusion: Based on the in vitro and in vivo investigations herein, functionalization of membranes with MSC‐CM represents a promising strategy to enhance GBR. [ABSTRACT FROM AUTHOR]

Objectives: Autologous bone is considered the gold standard for grafting, yet it suffers from a tendency to undergo resorption over time. While the exact mechanisms of this resorption remain elusive, osteocytes have been shown to play an important role in stimulating osteoclastic activity through their expression of receptor activator of NF‐κB (RANK) ligand (RANKL). The aim of this study was to assess the function of osteocyte‐derived RANKL in bone graft remodeling. Materials and Methods: In Tnfsf11fl/fl;Dmp1‐Cre mice without osteocyte‐specific RANKL as well as in Dmp1‐Cre control mice, 2.6 mm calvarial bone disks were harvested and transplanted into mice with matching genetic backgrounds either subcutaneously or subperiosteally, creating 4 groups in total. Histology and micro‐computed tomography of the grafts and the donor regions were performed 28 days after grafting. Results: Histology revealed marked resorption of subcutaneous control Dmp1‐Cre grafts and new bone formation around subperiosteal Dmp1‐Cre grafts. In contrast, Tnfsf11fl/fl;Dmp1‐Cre grafts showed effectively neither signs of bone resorption nor formation. Quantitative micro‐computed tomography revealed a significant difference in residual graft area between subcutaneous and subperiosteal Dmp1‐Cre grafts (p <.01). This difference was not observed between subcutaneous and subperiosteal Tnfsf11fl/fl;Dmp1‐Cre grafts (p =.17). Residual graft volume (p =.08) and thickness (p =.13) did not differ significantly among the groups. Donor area regeneration was comparable between Tnfsf11fl/fl;Dmp1‐Cre and Dmp1‐Cre mice and restricted to the defect margins. Conclusions: The results suggest an active function of osteocyte‐derived RANKL in bone graft remodeling. [ABSTRACT FROM AUTHOR]

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel 'off-the-shelf' strategy for GBR. [ABSTRACT FROM AUTHOR]

A static magnetic field generated by neodymium–iron–boron (NdFeB) magnets placed in the inner cavity of dental implants can enhance bone regeneration in rabbits. It is, however, unknown whether static magnetic fields support osseointegration in a canine model. We therefore determined the potential osteogenic effect of implants carrying NdFeB magnets inserted in the tibia of six adult canines in the early stages of osseointegration. Here, we report that after 15 days of healing, magnetic and regular implants showed a high variation with a median new bone-to-implant contact (nBIC) in the cortical (41.3% and 7.3%) and the medullary (28.6% and 44.8%) region, respectively. Consistently, the median new bone volume/tissue volume (nBV/TV) in the cortical (14.9% and 5.4%) and the medullary (22.2% and 22.4%) region were not significantly different. One week of healing only resulted in negligible bone formation. These findings suggest that considering the large variation and the pilot nature of this study, magnetic implants failed to support peri-implant bone formation in a canine model. [ABSTRACT FROM AUTHOR]

During bone development, FasL acts not only through the traditional apoptotic mechanism regulating the amount of bone-resorbing osteoclasts, but there is also growing evidence about its effect on cell differentiation. Expression of osteoblastic factors was followed in non-differentiated and differentiating primary calvarial cells obtained from FasL-deficient (gld) mice. The gld cells showed decreased expression of the key osteoblastic molecules osteocalcin (Ocn), osteopontin (Opn), and alkaline phosphatase (Alpl) in both groups. Notably, receptor activator of nuclear factor kappa-B ligand (Rankl) was unchanged in non-differentiated gld vs. wild type (wt) cells but decreased in differentiating gld cells. Osteoprotegerin (Opg) in the gld samples was increased in both groups. Opg vs. Rankl expression levels favored Opg in the case of non-differentiated cells but Rankl in differentiating ones. These results expand information on the involvement of FasL in non-apoptotic cell pathways related to osteoblastogenesis and consequently also osteoclastogenesis and pathologies such as osteoporosis. [ABSTRACT FROM AUTHOR]

Simvastatin embedded into poly(lactic-co-glycolic acid) (PLGA)-based scaffolds can stimulate bone regeneration in preclinical models. However, the ideal pharmacological dose has not been evaluated. This systematic review reports on the simvastatin doses used in preclinical studies and evaluates the regeneration of critical-sized bone defects. References were selected in a two-phase process. Electronic databases (Embase, LILACS, LIVIVO, PubMed, SCOPUS, and Web of Science) and grey literature databases (Google Scholar, Open Grey, and ProQuest) were searched until September 2022. The risk of bias was considered to be low based on the SYRCLE tool. We identified four studies in rat, two in parietal and two in calvaria bone, one in mouse parietal bone, and one in rabbit femur bone. Simvastatin, ranging from 8 to 100 µg, significantly increased bone formation in five studies, as compared to the scaffold alone based on µ-computed tomography, histomorphometric, and radiography analysis. The median increase in bone formation caused by simvastatin was 2.1-fold compared to the PLGA-based scaffold alone. There was, however, no significant correlation between the relative bone gain and the doses of simvastatin (p = 0.37). The data suggest that relatively lower doses of simvastatin can consistently promote preclinical bone regeneration. However, the interpretation of these data must consider the heterogenicity of the PLGA-scaffolds, the defect anatomy, the observation period, and the evaluation method. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of platelet-rich fibrin on bone formation by investigating its use in guided bone regeneration, sinus elevation and implant therapy. Materials and methods: This systematic review and meta-analysis were conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The eligibility criteria comprised human controlled clinical trials comparing the clinical outcomes of platelet-rich fibrin with those of other treatment modalities. The outcomes measured included percentage of new bone formation, percentage of residual bone graft, implant survival rate, change in bone dimension (horizontal and vertical), and implant stability quotient values. Results: From 320 articles identified, 18 studies were included. Owing to the heterogeneity of the investigated parameters, a meta-analysis was only possible for sinus elevation. There is a general lack of data from comparative randomised clinical trials evaluating platelet-rich fibrin for guided bone regeneration procedures (only two studies), with no quantifiable advantages in terms of new bone formation or dimensional bone gain found in the platelet-rich fibrin group. For sinus elevation, the meta-analysis demonstrated no advantage in terms of histological new bone formation in the control group (bone graft alone) compared with the test group (bone graft and platelet-rich fibrin). Two studies demonstrated that platelet-rich fibrin may shorten healing periods prior to implant placement. Platelet-rich fibrin was also shown to slightly enhance primary implant stability (implant stability quotient value < 5) as assessed using implant stability quotients and resonance frequency analysis parameters, with no histological data evaluating bone-implant contact yet available on this topic. In one study, platelet-rich fibrin was shown to improve the clinical parameters when utilised as an adjunct for the treatment of peri-implantitis. Conclusions: In the majority of studies, platelet-rich fibrin offered little or no clear advantage in terms of new bone formation as evaluated in various studies on guided bone regeneration and sinus elevation, nor in implant stability and treatment of peri-implantitis. Various authors and systematic reviews on the topic have now expressed criticism of the various study designs and protocols, and the lack of appropriate controls and available information regarding patient selection. Well-controlled human studies on these specific topics are required. [ABSTRACT FROM AUTHOR]

Background: Bilayer collagen membranes are routinely used in guided bone/tissue regeneration to serve as osteoconductive scaffolds and prevent the invasion of soft tissues. It is recommended to place the membranes with their dense layer towards the soft tissue and their porous layer towards the bony defect area. However, evidence supporting this recommendation is lacking. This study aimed to determine whether the alignment of bilayer collagen membranes has an effect on bone regeneration. Methods: In two groups of ten male Sprague-Dawley rats each, a 5-mm calvarial defect was created. Thereafter, the defect was randomly covered with a bilayer, resorbable, pure type I and III collagen membrane placed either regularly or upside-down (i.e., dense layer towards bone defect). After 4 weeks of healing, micro-computed tomography (μCT), histology, and histomorphometry of the inner cylindrical region of interest (4.5 mm in diameter) were performed to assess new bone formation and the consolidation of the collagen membrane in the defect area. Results: Quantitative μCT showed similar bone volume (median 8.0 mm3, interquartile range 7.0–10.0 vs. 6.2 mm3, 4.3–9.4, p = 0.06) and trabecular thickness (0.21 mm, 0.19–0.23 vs. 0.18 mm, 0.17–0.20, p = 0.03) between upside-down and regular placement, both leading to an almost complete bony coverage. Histomorphometry showed comparable new bone areas between the upside-down and regularly placed membranes, 3.9 mm2 (2.7–5.4) vs. 3.8 mm2 (2.2–4.0, p = 0.31), respectively. Both treatment groups revealed the same regeneration patterns and spatial distribution of bone with and without collagen fibers, as well as residual collagen fibers. Conclusions: Our data support the osteoconductive properties of collagen membranes and suggest that bone regeneration is facilitated regardless of membrane layer alignment. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the use of platelet-rich fibrin for alveolar ridge preservation compared to natural healing, bone graft material and platelet-rich fibrin in combination with bone graft material. Materials and methods: The present systematic review was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis guidelines. The review examined randomised controlled trials comparing the clinical outcomes of platelet-rich fibrin with those of other modalities for alveolar ridge preservation. Studies of third molar extraction site healing were excluded. The studies were classified into three categories: natural wound healing vs platelet-rich fibrin; bone graft material vs platelet-rich fibrin; and bone graft material vs bone graft material and platelet-rich fibrin. Results: From 179 articles identified, 16 randomised controlled trials were included. Owing to the heterogeneity of the investigated parameters, it was not possible to perform a meta-analysis. In total, 10 randomised controlled trials compared platelet-rich fibrin to natural wound healing, with seven of these demonstrating favourable outcomes to either limit postextraction dimensional changes or improve new bone formation in the platelet-rich fibrin group. Three of four studies comparing healing with bone graft material to platelet-rich fibrin found that the latter led to significantly greater horizontal or vertical bone resorption, and the bone graft material was more able to maintain the ridge dimensions. Two out of three randomised controlled trials investigating healing with both bone graft material and platelet-rich fibrin reported better outcomes using this combined approach than with bone graft material alone. All studies investigating soft tissue healing with platelet-rich fibrin demonstrated better outcomes in the platelet-rich fibrin group. Conclusions: The majority of studies comparing healing with platelet-rich fibrin to natural healing concluded that the former more successfully limits postextraction dimensional changes than the latter. However, 75% of studies investigating platelet-rich fibrin vs bone graft material reported better results in the bone graft group with respect to its ability to maintain postextraction dimensional changes. The addition of platelet-rich fibrin to bone graft material may improve clinical outcomes, although data are limited. [ABSTRACT FROM AUTHOR]

Background: Inflammatory osteolysis is the clinical hallmark of peri-implantitis. The morphology of the remaining peri-implant bone and the level of osseointegration, however, remain unknown. Our aim was to characterize advanced peri-implantitis bone defects in humans. Methods: Four patients (3 female and 1 male) were diagnosed with peri-implantitis. A total of 5 implants with machined surfaces and a mean loading time of 12 ± 6 years were removed due to advanced bone loss. The defect extension, the peri-implant bone density (bone area per tissue area in percentage), bone-to-implant contact (%), and the number of filled and empty osteocyte lacunae were calculated based on undecalcified histological specimens. Results: The defect extension was on average 4.2 mm (95% CI 0.8–3.4). Remaining peri-implant bone showed a high density of 85.5% (95% CI 79.1–91.3) and covered in total 74% (95% CI 70.5–77.5) of the implant surface. Filled and empty osteocyte lacunae density was on average 191 and 165/mm2 (95% CI 132–251; 103–225), respectively. Histology further revealed signs of ongoing bone formation and resorption. Conclusion: There are signs that suggest that once the original cortical bone is lost due to peri-implantitis, the remaining apical trabecular bone is reinforced and transformed into cortical bone that might take over the functional load. [ABSTRACT FROM AUTHOR]

Background: Platelet-rich fibrin (PRF) membranes can preserve alveolar ridge dimension after tooth extraction. Thus, it can be presumed that PRF suppresses the catabolic events that are caused by osteoclastic bone resorption.Methods: To address this possibility, we investigated the impact of soluble extracts of PRF membranes on in vitro osteoclastogenesis in murine bone marrow cultures. Osteoclastogenesis was induced by exposing murine bone marrow cultures to receptor activator of nuclear factor kappa B ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and transforming growth factor-beta 1 (TGF-β1) in the presence or absence of PRF. Osteoclastogenesis was evaluated based on histochemical, gene expression, and resorption analysis. Viability was confirmed by formation of formazan crystals, live-dead staining and caspase-3 activity assay.Results: We report here that in vitro osteoclastogenesis is greatly suppressed by soluble extracts of PRF membranes as indicated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation. In support of the histochemical observations, soluble extracts of PRF membranes decreased expression levels of the osteoclast marker genes TRAP, Cathepsin K, dendritic cell-specific transmembrane protein (DCSTAMP), nuclear factor of activated T-cells (NFATc1), and osteoclast-associated receptor (OSCAR). PRF membranes, however, cannot reverse the process once osteoclastogenesis has evolved.Conclusion: These in vitro findings indicate that PRF membranes can inhibit the formation of osteoclasts from hematopoietic progenitors in bone marrow cultures. Overall, our results imply that the favorable effects of PRF membranes in alveolar ridge preservation may be attributed, at least in part, by the inhibition of osteoclastogenesis. [ABSTRACT FROM AUTHOR]

Aim: Osteoimmunology covers the cellular and molecular mechanisms responsible for inflammatory osteolysis that culminates in the degradation of alveolar bone. Osteoimmunology also focuses on the interplay of immune cells with bone cells during bone remodelling and regeneration. The aim of this review was to provide insights into how osteoimmunology affects alveolar bone health and disease. Method: This review is based on a narrative approach to assemble mouse models that provide insights into the cellular and molecular mechanisms causing inflammatory osteolysis and on the impact of immune cells on alveolar bone regeneration. Results: Mouse models have revealed the molecular pathways by which microbial and other factors activate immune cells that initiate an inflammatory response. The inflammation‐induced alveolar bone loss occurs with the concomitant suppression of bone formation. Mouse models also showed that immune cells contribute to the resolution of inflammation and bone regeneration, even though studies with a focus on alveolar socket healing are rare. Conclusions: Considering that osteoimmunology is evolutionarily conserved, osteolysis removes the cause of inflammation by provoking tooth loss. The impact of immune cells on bone regeneration is presumably a way to reinitiate the developmental mechanisms of intramembranous and endochondral bone formation. [ABSTRACT FROM AUTHOR]

Background and Aims: To describe the biology of alveolar bone regeneration. Material and Methods: Four comprehensive reviews were performed on (a) mesenchymal cells and differentiation factors leading to bone formation; (b) the critical interplay between bone resorbing and formative cells; (c) the role of osteoimmunology in the formation and maintenance of alveolar bone; and (d) the self‐regenerative capacity following bone injury or tooth extraction were prepared prior to the workshop. Results and Conclusions: This summary information adds to the fuller understanding of the alveolar bone regenerative response with implications to reconstructive procedures for patient oral rehabilitation. The group collectively formulated and addressed critical questions based on each of the reviews in this consensus report to advance the field. The report concludes with identified areas of future research. [ABSTRACT FROM AUTHOR]

Background Current strategies to reduce medical device-associated infections propose zirconia as a potential implant material which may limit bacterial adhesion. Because multinucleated giant cells ( MNGCs) have been detected on these implant surfaces, concerns have been raised regarding tissue integration. Purpose The present study examined the presence of MNGCs and their subsequent effect upon tissue integration. Surface-modified implants made of yttria-stabilized ( TZP) and alumina-toughened zirconia ( ATZ) were compared with commercially pure titanium ( Ti). Materials and Methods Seven miniature pigs received three implants on either side of the maxilla. After healing periods of 4 and 8 weeks, the tissue response at the implant surfaces was characterized according to three specific parameters: bone-to-implant contact ( BIC), MNGC-to-implant contact ( MIC), and the peri-implant bone density ( BD). Results Despite being present on all tested implant surfaces, MNGCs were not associated with an inflammatory cell infiltrate or with fibrous encapsulation. MNGCs were less numerous on the Ti implants (range: 3.9-5.2%) compared with the ceramic implants (range: 17.6-30.3%, p < .0001). Even though the values of newly formed bone and pristine bone in direct contact with the implant surfaces were high at 4 weeks ( tBIC: Ti = 82.3%, TZP = 64.3%, ATZ = 70%), a negative correlation was observed between the presence of MNGCs and newly formed bone at the implant surface ( p < .001). Interestingly, the newly formed peri-implant bone density, defined as the percentage of new bone area inside the screw threads ( nBD), was not diminished by the presence of MNGCs. Conclusions Differences in the presence of MNGCs and the BIC parameters between Ti and the ceramic implants appear to be a local cellular phenomenon which is restricted to the implant-bone marrow interface and do not affect the peri-implant bone formation. Factors triggering MNGC differentiation and their persistence in response to biomaterial surface need to be investigated in future studies. [ABSTRACT FROM AUTHOR]

Objectives Since the original description of osteoinduction in the early 20th century, the study and development of innovative biomaterials has emerged. Recently, novel synthetic bone grafts have been reported with potential to form ectopic bone in vivo. However, their full characterization in comparison with other leading bone grafts has not been investigated. The aim of this study was to determine the osteoinductive potential of bone grafts by comparing autogenous bone grafts, demineralized freeze-dried bone allografts ( DFDBA), a commonly utilized natural bone mineral ( NBM) from bovine origin (Bio-Oss), and a newly developed biphasic calcium phosphate ( BCP). Materials and methods Grafts were compared in vitro for their ability to stimulate bone marrow stromal cell ( BMSC) migration, proliferation, and differentiation as assessed by quantitative real-time PCR for genes coding for bone markers including Runx2, collagen I, and osteocalcin. Furthermore, bone grafts were implanted in the calf muscle of 12 beagle dogs to determine their potential to form ectopic bone in vivo. Results The in vitro results demonstrate that both autografts and DFDBA show potential for cell recruitment, whereas only autografts and BCP demonstrated the ability to differentiate BMSCs toward the osteoblast lineage. The in vivo ectopic bone model demonstrated that while NBM particles were not osteoinductive and autogenous bone grafts were resorbed quickly in vivo, ectopic bone formation was reported in DFDBA and in synthetic BCP grafts. Conclusion The modifications in nanotopography and chemical composition of the newly developed BCP bone grafts significantly promoted ectopic bone formation confirming their osteoinductive potential. In conclusion, the results from this study provide evidence that synthetic bone grafts not only serve as a three-dimensional scaffold but are also able to promote osteoinduction. [ABSTRACT FROM AUTHOR]

Background: Supplementation of bone substitutes with recombinant platelet-derived growth factor-BB (PDGF-BB) can enhance bone regeneration. The aim of the study was to evaluate the effect of PDGF-BB on bone formation in the presence of β-tricalcium phosphate and bovine bone mineral matrix in a rat calvaria defect model. Methods: The authors examined 5 mm rat calvarial defects treated with β-tricalcium phosphate (TCP) or demineralized bovine bone mineral (DBBM) with and without 0.3 mg/ml recombinant PDGF-BB. Calvaria defects were randomly divided into the following treatment groups (n = 5); TCP; TCP plus PDGF-BB; DBBM; DBBM plus PDGF-BB; and untreated empty control. After 45 days, bone formation was evaluated by histomorphometry and fluorescence microscopy. Results: The authors report that the area of newly formed bone was similar between the empty controls and the two bone substitutes, TCP and DBBM. Supplementation of TCP and DBBM with PDGF-BB had no significant impact on bone formation. Fluorochrome staining revealed no visible changes in the pattern of bone formation in defects filled with TCP and DBBM, irrespective of PDGF-BB. Furthermore, supplementation with PDGF-BB did not influence biomaterial degradation. Conclusions: The authors concluded that PDGF-BB had no impact on bone formation and degradation of bone substitutes in the respective rodent models. Thus, possible beneficial effects of PDGF-BB may require other model situations. Keywords: Bone substitutes, Bone formation, Bone regeneration, Bone augmentation, Platelet-derived growth factor-BB, Rat calvaria defects, β-tricalcium phosphate, Demineralized bovine bone mineral, Histomorphometry, Graft consolidation [ABSTRACT FROM AUTHOR]

Background: Preclinical studies support the assumption that connective tissue grafts preserve the alveolar bone from resorption; the underlying cellular mechanisms, however, remain unknown. The cellular mechanisms may be attributed to the paracrine activity of the palatal fibroblasts. It was thus reasonable to suggest that palatal connective tissue grafts reduce the formation of osteoclasts. Methods: To test this hypothesis, human palatal fibroblasts were examined for their capacity to modulate the formation of osteoclasts in murine bone marrow cultures exposed to RANKL, M-CSF and TGF-β1. Osteoclastogenesis was determined by tartrate-resistant acid phosphatase (TRAP) staining and gene expression analysis. The formation of antigen presenting cells was based on the expression of CD14 and costimmulatory molecules of antigen presenting cells. The paracrine interaction of fibroblasts and the bone marrow was modeled in vitro with inserts of cell-occlusive membranes. Results: In cocultures without cell-to-cell contact, palatal fibroblasts caused a decrease in the expression of the osteoclast marker genes in bone marrow cells; calcitonin receptors, cathepsin K, TRAP, and osteoclast-associated receptor. Also the number of TRAP positive multinucleated cells was decreased in the presence of fibroblasts. Notably, palatal fibroblasts increased the expression of CD14 and the co-stimulatory proteins CD40, CD80, and CD86 in bone marrow cells. Bone marrow cells had no considerable impact on fibroblast viability and proliferation marker genes. With regard to cell distribution, osteoclasts were most prominent in the center of the membranes, while fibroblasts accumulated immediately adjacent to the border of the insert forming a ring-like structure on the surface of the culture plate. Conclusion: The data suggest that palatal fibroblasts provide a paracrine environment that reduces osteoclastogenesis and increases markers of antigen presenting cells. Morover, the paracrine model revealed a joint activity between palatal fibroblasts and bone marrow cells visualized by the characteristic cell distribution in the two separated compartments. [ABSTRACT FROM AUTHOR]

Background and Purpose Autografts are used for bone reconstruction in regenerative medicine including oral and maxillofacial surgery. Bone grafts release paracrine signals that can reach mesenchymal cells at defect sites. The impact of the paracrine signals on osteogenic, adipogenic, and chondrogenic differentiation of mesenchymal cells has remained unclear. Material and Methods Osteogenesis, adipogenesis, and chondrogenesis were studied with murine ST2 osteoblast progenitors, 3 T3- L1 preadipocytes, and ATDC5 prechondrogenic cells, respectively. Primary periodontal fibroblasts from the gingiva, from the periodontal ligament, and from bone were also included in the analysis. Cells were exposed to bone-conditioned medium ( BCM) that was prepared from porcine cortical bone chips. Results BCM inhibited osteogenic and adipogenic differentiation of ST2 and 3 T3- L1 cells, respectively, as shown by histological staining and gene expression. No substantial changes in the expression of chondrogenic genes were observed in ATDC5 cells. Primary periodontal fibroblasts also showed a robust decrease in alkaline phosphatase and peroxisome proliferator-activated receptor gamma ( PPARγ) expression when exposed to BCM. BCM also increased collagen type 10 expression. Pharmacologic blocking of transforming growth factor ( TGF)-β receptor type I kinase with SB431542 and the smad-3 inhibitor SIS3 at least partially reversed the effect of BCM on PPARγ and collagen type 10 expression. In support of BCM having TGF-β activity, the respective target genes were increasingly expressed in periodontal fibroblasts. Conclusions The present work is a pioneer study on the paracrine activity of bone grafts. The findings suggest that cortical bone chips release soluble signals that can modulate differentiation of mesenchymal cells in vitro at least partially involving TGF-β signaling. [ABSTRACT FROM AUTHOR]

Objectives: Uncontrolled diabetes mellitus is associated with impaired osseointegration. Diabetic individuals might benefit from bone anabolic therapies. Intermittent administration of 1-34 parathyroid hormone (PTH) stimulates bone formation in rodent models. However, this anabolic effect fails in diabetic rats. Whether the anabolic effect of PTH can be achieved in insulincontrolled diabetic rats has not been investigated yet. Materials and Methods: After diabetes induction with streptozotocin in 40 female Wistar rats, the animals were randomly divided into 4 groups: diabetes, diabetes plus PTH, insulin-treated diabetes, and insulin-treated diabetes plus PTH. After 1 week, miniscrews were inserted in the tibiae. Osmotic pumps with insulin or saline solution were implanted. Animals received 60 mg/kg PTH or saline solution. Histomorphometric analysis was performed. Results: In diabetic rats, no changes of medullary periimplant bone area or bone-to-implant contacts (BICs) were achieved with or without treatment with PTH. However, also animals treated with insulin failed to response significantly to PTH regarding bone area (7.4 ± 4.1% and 8.1 ± 4.1%) and BICs (33.7 ± 16.9% and 49.9 ± 11.9%). Conclusion: These results demonstrate that the metabolic characteristics of the diabetic rats produced a condition unable to respond to PTH treatment, even when hyperglycemia was controlled with insulin. [ABSTRACT FROM AUTHOR]

Background Harvesting techniques can affect cellular parameters of autogenous bone grafts in vitro. Whether these differences translate to in vivo bone formation, however, remains unknown. Objective The purpose of this study was to assess the impact of different harvesting techniques on bone formation and graft resorption in vivo. Material and methods Four harvesting techniques were used: (i) corticocancellous blocks particulated by a bone mill; (ii) bone scraper; (iii) piezosurgery; and (iv) bone slurry collected from a filter device upon drilling. The grafts were placed into bone defects in the mandibles of 12 minipigs. The animals were sacrificed after 1, 2, 4 and 8 weeks of healing. Histology and histomorphometrical analyses were performed to assess bone formation and graft resorption. An explorative statistical analysis was performed. Results The amount of new bone increased, while the amount of residual bone decreased over time with all harvesting techniques. At all given time points, no significant advantage of any harvesting technique on bone formation was observed. The harvesting technique, however, affected bone formation and the amount of residual graft within the overall healing period. Friedman test revealed an impact of the harvesting technique on residual bone graft after 2 and 4 weeks. At the later time point, post hoc testing showed more newly formed bone in association with bone graft processed by bone mill than harvested by bone scraper and piezosurgery. Conclusions Transplantation of autogenous bone particles harvested with four techniques in the present model resulted in moderate differences in terms of bone formation and graft resorption. [ABSTRACT FROM AUTHOR]

Objectives Osteocytes, the most common cells of the bone, are buried in lacunae. Density and area of the osteocyte lacunae change with increasing maturation of the newly formed bone. Evaluation of osteocyte lacunae can therefore provide insights into the process of graft consolidation. Materials and Methods Here, we determined the osteocyte lacunar density (number of osteocyte lacunae per bone area; N.Ot/ BAr) and the osteocyte lacunar area in μm2 (Lac.Ar) in histological specimens 6 and 12 weeks after the sinuses of 10 minipigs were augmented with Bio-Oss®, a deproteinized bovine bone mineral, and Ostim®, an aqueous paste of synthetic nanoparticular hydroxyapatite. The region of interest was defined by the following criteria: (i) >1 mm from the host bone, (ii) >0.5 mm from the sinus mucosa, (iii) minimum area of 0.2 mm2, and (iv) bone tissue spanning at least two bone substitute particles. Results The overall osteocyte lacunar density was significantly higher in the Bio-Oss® group than in the Ostim® group and decreased during the observation period at a similar range in both groups. The osteocyte lacunar area was smaller in the Bio-Oss® group than the Ostim® group but there was no significant change within the groups over time. Conclusions These results suggest that bone substitutes affect the osteocyte lacunar density and the osteocyte lacunar area in the newly formed bone within the augmented sinus in this particular model situation. These measures can provide insights into the maturation of newly formed bone in the augmented sinus. [ABSTRACT FROM AUTHOR]

There is a need for evaluating zirconia surface modifications and their potential impact on the biological response of osteogenic cells. Grit blasted zirconia discs were either left untreated or underwent acid or alkaline etching. Adhesion and osteogenic differentiation of MG63 cells was determined after one week of culture. The macro-scaled roughness of the grit blasted zirconia discs, independent of the surface treatment, was within a narrow range and only slightly smoother than titanium discs. However, the alkaline- and acid-etching led to an increase of the micro-roughness of the surface. The surface modifications had no effect on cell spreading and did not cause significant change in the expression of differentiation markers. Thus, in this respective setting, morphologic changes observed upon treatment of grit blasted zirconia discs with acid or alkaline do not translate into changes in MG63 cell adhesion or differentiation and are comparable to findings with anodized titanium discs. [ABSTRACT FROM AUTHOR]

The aim of thisin vitrostudy was to determine whether the sinus mucosa holds cells with an osteogenic potential. Frozen sections of sinus mucosa from three adult pigs were investigated for the expression of STRO-1, a marker of mesenchymal progenitor cells, and alkaline phosphatase activity, an enzyme expressed by cells committed to the osteogenic lineage and by mature osteoblasts. To determine their osteogenic potential, mucosa-derived cells were incubated with bone morphogenetic protein (BMP)-6 and BMP-7, and alkaline phosphatase activity, osteocalcin expression, and mineralization of the extracellular matrix was measured. We found sinus mucosa cells staining positive for STRO-1 and alkaline phosphatase activity. When sinus mucosa tissue was placed in culture, alkaline phosphatase positive cells grew out from the explants and further increased alkaline phosphatase activity in response to BMP-6 and BMP-7. The expression level of the osteoblast-specific extracellular matrix protein osteocalcin, and the amount of calcium accumulation within the extracellular matrix was also increased in response to BMPs. We conclude that the sinus mucosa holds mesenchymal progenitor cells and cells committed to the osteogenic lineage that can respond to BMP-6 and BMP-7 by an increase of their osteogenic differentiation.To cite this article:Gruber R, Kandler B, Fuerst G, Fischer MB, Watzek G. Porcine sinus mucosa holds cells that respond to bone morphogenetic protein (BMP)-6 and BMP-7 with increased osteogenic differentiationin vitro.Clin. Oral Impl. Res.doi: 10.1111/j.1600-0501.2004.01062.x [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: FasL used to be considered as a classical ligand triggering cell death (apoptosis) via its receptor, Fas and thefollowing caspase cascade. As such, it is known to be involved in regulation within the bone. Recently, however, the knowledge has expanded about the non-apoptotic and caspase-independent engagement of the Fas/FasL pathway. The present investigation identified that stimulation of osteocytic IDG-SW3 cells by FasL leads to a dramatic decrease in expression of the major osteocytic marker, sclerostin. Additionally, other key components of the osteogenic pathways were impacted, notably in a caspase-independent manner. Such findings are of importance for basic biology as well as biomedical applications since osteocytes are the major population within adult bones and Fas signalling is one of therapeutical targets, e.g., in the anti-osteoporotic treatment. The Fas ligand (FasL) is known from programmed cell death, the immune system, and recently also from bone homeostasis. As such, Fas signalling is a potential target of anti-osteoporotic treatment based on the induction of osteoclastic cell death. Less attention has been paid to osteocytes, although they represent the majority of cells within the mature bone and are the key regulators. To determine the impact of FasL stimulation on osteocytes, differentiated IDG-SW3 cells were challenged by FasL, and their osteogenic expression profiles were evaluated by a pre-designed PCR array. Notably, the most downregulated gene was the one for sclerostin, which is the major marker of osteocytes and a negative regulator of bone formation. FasL stimulation also led to significant changes (over 10-fold) in the expression of other osteogenic markers: Gdf10, Gli1, Ihh, Mmp10, and Phex. To determine whether these alterations involved caspase-dependent or caspase-independent mechanisms, the IDG-SW3 cells were stimulated by FasL with and without a caspase inhibitor: Q-VD-OPh. The alterations were also detected in the samples treated by FasL along with Q-VD-OPh, pointing to the caspase-independent impact of FasL stimulation. These results contribute to an understanding of the recently emerging pleiotropic effects of Fas/FasL signalling and specify its functions in bone cells. [ABSTRACT FROM AUTHOR]

Volume-stable collagen matrices (VSCM) are conductive for the connective tissue upon soft tissue augmentation. Considering that collagen has osteoconductive properties, we have investigated the possibility that the VSCM also consolidates with the newly formed bone. To this end, we covered nine rat calvaria circular defects with a VSCM. After four weeks, histology, histomorphometry, quantitative backscattered electron imaging, and microcomputed tomography were performed. We report that the overall pattern of mineralization inside the VSCM was heterogeneous. Histology revealed, apart from the characteristic woven bone formation, areas of round-shaped hypertrophic chondrocyte-like cells surrounded by a mineralized extracellular matrix. Quantitative backscattered electron imaging confirmed the heterogenous mineralization occurring within the VSCM. Histomorphometry found new bone to be 0.7 mm2 (0.01 min; 2.4 max), similar to the chondrogenic mineralized extracellular matrix with 0.7 mm2 (0.0 min; 4.2 max). Microcomputed tomography showed the overall mineralized tissue in the defect to be 1.6 mm3 (min 0.0; max 13.3). These findings suggest that in a rat cranial defect, VSCM has a limited and heterogeneous capacity to support intramembranous bone formation but may allow the formation of bone via the endochondral route. [ABSTRACT FROM AUTHOR]

Simple Summary: Tooth roots are increasingly applied for bone reconstruction before implant placement. Growth factors stored in the dentin are assumed to enhance bone regeneration, however, the evidence is low. To this aim, collagen membranes were coated with dentin lysates obtained from extracted porcine teeth or remain untreated. The collagen membranes were tested for their capacity to stimulate bone formation in rat calvarial bone defects. After four weeks of healing, micro-computed tomography and histological analyses revealed that dentin lysates coating had no significant impact on the rather strong bone regeneration reaching a nearly complete defect closure even in untreated defects. It can thus be concluded that dentin lysates do not hinder bone regeneration. Conclusions concerning a possible stimulation of bone regeneration by dentin lysates should not be drawn. Autogenous tooth roots are increasingly applied as a grafting material in alveolar bone augmentation. Since tooth roots undergo creeping substitution similar to bone grafts, it can be hypothesized that osteoclasts release the growth factors stored in the dentin thereby influencing bone formation. To test this hypothesis, collagen membranes were either soaked in acid dentin lysates (ADL) from extracted porcine teeth or serum–free medium followed by lyophilization. Thereafter, these membranes covered standardized 5-mm-diameter critical-size defects in calvarial bone on rats. After four weeks of healing, micro-computed tomography and histological analyses using undecalcified thin ground sections were performed. Micro-computed tomography of the inner 4.5 mm calvaria defects revealed a median bone defect coverage of 91% (CI: 87–95) in the ADL group and 94% (CI: 65–100) in the control group, without significant differences between the groups (intergroup p > 0.05). Furthermore, bone volume (BV) was similar between ADL group (5.7 mm3, CI: 3.4–7.1) and control group (5.7 mm3, CI: 2.9–9.7). Histomorphometry of the defect area confirmed these findings with bone area values amounting to 2.1 mm2 (CI: 1.2–2.6) in the ADL group and 2.0 mm2 (CI: 1.1–3.0) in the control group. Together, these data suggest that acid dentin lysate lyophilized onto collagen membranes failed to modulate the robust bone formation when placed onto calvarial defects. [ABSTRACT FROM AUTHOR]

Background: Demineralized bone matrix is a widely used allograft from where not only the inorganic mineral but also embedded growth factors are removed by 0.1 M hydrochloric acid. The cellular response to the growth factors released during the preparation of demineralized bone matrix has not been studied. Aim/Hypothesis: To investigate the in vitro impact of acid bone lysates (ABL) prepared from porcine cortical bone chips on oral fibroblasts. Material and Methods: Whole genome microarray was performed to screen for the ABL target genes in oral fibroblasts. Gene expression changes were confirmed by RT‐PCR and the translation into protein done by immunoassay. Immunostaining was implemented to visualize nuclear accumulation of Smad‐3. Results: Whole genome microarray together with the pharmacologic blocking of TGF‐β receptor type I kinase with SB431542 revealed that acid bony lysates activate TGF‐β signaling in oral fibroblasts. These findings were further confirmed by the SB431542‐dependent expression of interleukin 11, proteoglycan4 and NADPH oxidase 4 in human oral fibroblasts and by the expression of interleukin 11 in pig periosteal fibroblasts. Interleukin 11 expression was confirmed at the protein level by immunoassay. In support of TGF‐β receptor type I kinase signaling, ABL activated the nuclear accumulation of Smad‐3. The effect was independent of ABL source, mandible, calvaria or tibia. Gene expression was, however, dependent on the concentration of hydrochloric acid. ABL prepared by 0.1 M HCL was most effective compared to lysates obtained with 0.01 M and also 1.0 M HCl. Furthermore, ABL inhibited expression of osteogenic but increased chondrogenic genes. Conclusions and Clinical Implications: In summary, these results demonstrate that TGF‐β is a major growth factor being released upon preparation of demineralized bone matrix. [ABSTRACT FROM AUTHOR]