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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]

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]

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]

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]

(1) Background: Inhibition of osteoclast differentiation is the key approach in treating osteoporosis. However, using state-of-the-art treatments such as bisphosphonates and estrogen-based therapy is usually accompanied by many side effects. As opposed to this, the use of natural products as an osteoporotic remedy delivers promising outcomes with minimal side effects. (2) Methods: In the present study, we implemented a biochemometric workflow comprising (i) chemometric approaches using NMR and mass spectrometry and (ii) cell biological approaches using an osteoclast cytochemical marker (TRAP). The workflow serves as a screening tool to pursue potential in vitro osteoclast inhibitors. (3) Results: The workflow allowed for the selective isolation of two phenylpropanoids (coniferyl alcohol and sinapyl alcohol) from the fruits of neem tree (Azadirachta indica). These two isolated phenylpropanoids showed a very promising dose-dependent inhibition of osteoclast differentiation with negligible effects in terms of cell viability. (4) Conclusion: The presented workflow is an effective tool in the discovery of potential candidates for osteoclast inhibition from complex extracts. The used biochemometric approach saves time, effort and costs while delivering precise hints to selectively isolate bioactive constituents. [ABSTRACT FROM AUTHOR]

Objectives: Chronic liver disease increases the risk for periodontal disease and osteoporotic fractures, but its impacts on bone regeneration remain unknown. Herein, we studied the impact of liver cirrhosis on peri‐implant bone formation. Material and Methods: A total of 20 male Wistar rats were randomly divided into two groups: one with the common bile duct ligated (BDL) and the respective sham‐treated control group (SHAM). After four weeks of disease induction, titanium mini‐screws were inserted into the tibia. Successful induction of liver cirrhosis was confirmed by the presence of clinical symptoms. Another four weeks later, peri‐implant bone volume per tissue volume (BV/TV) and bone‐to‐implant contact (BIC) were determined by histomorphometric analysis. Results: Peri‐implant bone formation was not significantly different between the SHAM and BDL groups. In the cortical compartment, the median percentage of peri‐implant new bone was 10.1% (95% CI of mean 4.0–35.7) and 22.5% (13.8–30.6) in the SHAM and BDL groups, respectively (p =.26). Consistently, the new bone in direct contact with the implant was 18.1% (0.4–37.8) and 23.3% (9.2–32.8) in SHAM and BDL groups, respectively (p =.38). When measuring the medullary compartment, the new bone area was 7.1% (4.8–10.4) and 10.4% (7.2–13.5) in the SHAM and BDL groups, respectively (p =.17). Medullary new bone in direct contact with the implant was 10.0% (1.2–50.4) and 20.6% (16.8–35.3) in SHAM and BDL groups, respectively, and thus comparable between the two groups (p =.46). Conclusions: Bile duct ligation has no significant impact on the early stages of peri‐implant bone formation. [ABSTRACT FROM AUTHOR]

Although autografts are considered to be the gold standard treatment for reconstruction of large bone defects resulting from trauma or diseases, donor site morbidity and limited availability restrict their use. Successful bone repair also depends on sufficient vascularization and to address this challenge, novel strategies focus on the development of vascularized biomaterial scaffolds. This pilot study aimed to investigate the feasibility of regenerating large bone defects in sheep using 3D-printed customized calcium phosphate scaffolds with or without surgical vascularization. Pre-operative computed tomography scans were performed to visualize the metatarsus and vasculature and to fabricate customized scaffolds and surgical guides by 3D printing. Critical-sized segmental defects created in the mid-diaphyseal region of the metatarsus were either left empty or treated with the 3D scaffold alone or in combination with an axial vascular pedicle. Bone regeneration was evaluated 1, 2 and 3 months post-implantation. After 3 months, the untreated defect remained non-bridged while the 3D scaffold guided bone regeneration. The presence of the vascular pedicle further enhanced bone formation. Histology confirmed bone growth inside the porous 3D scaffolds with or without vascular pedicle inclusion. Taken together, this pilot study demonstrated the feasibility of precised pre-surgical planning and reconstruction of large bone defects with 3D-printed personalized scaffolds. [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]

Platelet-rich fibrin, the coagulated plasma fraction of blood, is commonly used to support natural healing in clinical applications. The rat calvaria defect is a standardized model to study bone regeneration. It remains, however, unclear if the rat calvaria defect is appropriate to investigate the impact of human PRF (Platelet-Rich Fibrin) on bone regeneration. To this end, we soaked Bio-Gide® collagen membranes in human or rat liquid concentrated PRF before placing them onto 5 mm calvarial defects in Sprague Dawley rats. Three weeks later, histology and micro-computed tomography (μCT) were performed. We observed that the collagen membranes soaked with rat PRF show the characteristic features of new bone and areas of mineralized collagen matrix, indicated by a median mineralized volume of 1.5 mm3 (range: 0.9; 5.3 mm3). Histology revealed new bone growing underneath the membrane and hybrid bone where collagen fibers are embedded in the new bone. Moreover, areas of passive mineralization were observed. The collagen membranes soaked with human PRF, however, were devoid of histological features of new bone formation in the center of the defect; only occasionally, new bone formed at the defect margins. Human PRF (h-PRF) caused a median bone volume of 0.9 mm3 (range: 0.3–3.3 mm3), which was significantly lower than what was observed with rat PRF (r-PRF), with a BV median of 1.2 mm3 (range: 0.3–5.9 mm3). Our findings indicate that the rat calvaria defect model is suitable for assessing the effects of rat PRF on bone formation, but caution is warranted when extrapolating conclusions regarding the efficacy of human PRF. [ABSTRACT FROM AUTHOR]

Background: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. Methods: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. Results: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. Conclusions: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics. [ABSTRACT FROM AUTHOR]

Background: Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects. The aim of this study was to compare the potential of human BMSC cultured as 2D monolayers or 3D spheroids encapsulated in constructs of 3D-printed poly-L-lactide-co-trimethylene carbonate scaffolds and modified human platelet lysate hydrogels (PLATMC-HPLG) for bone regeneration. Methods: PLATMC-HPLG constructs with 2D or 3D BMSC were assessed for osteogenic differentiation based on gene expression and in vitro mineralization. Subsequently, PLATMC-HPLG constructs with 2D or 3D BMSC were implanted in rat calvarial defects for 12 weeks; cell-free constructs served as controls. Bone regeneration was assessed via in vivo computed tomography (CT), ex vivo micro-CT and histology. Results: Osteogenic gene expression was significantly enhanced in 3D versus 2D BMSC prior to, but not after, encapsulation in PLATMC-HPLG constructs. A trend for greater in vitro mineralization was observed in constructs with 3D versus 2D BMSC (p > 0.05). In vivo CT revealed comparable bone formation after 4, 8 and 12 weeks in all groups. After 12 weeks, micro-CT revealed substantial regeneration in 2D BMSC (62.47 ± 19.46%), 3D BMSC (51.01 ± 24.43%) and cell-free PLATMC-HPLG constructs (43.20 ± 30.09%) (p > 0.05). A similar trend was observed in the histological analysis. Conclusion: Despite a trend for superior in vitro mineralization, constructs with 3D and 2D BMSC performed similarly in vivo. Regardless of monolayer or spheroid cell culture, PLATMC-HPLG constructs represent promising scaffolds for bone tissue engineering applications. [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]