Human versus Rat PRF on Collagen Membranes: A Pilot Study of Mineralization in Rat Calvaria Defect Model.

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.

Active and Passive Mineralization of Bio-Gide® Membranes in Rat Calvaria Defects.

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.

Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

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.

The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet-rich fibrin.

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.

Lateral angle: A landmark-based method for the sex estimation in human cremated remains and application to an Austrian prehistoric sample.

OBJECTIVES: Estimating the sex of cremated human remains is difficult. The petrous bone frequently survives the cremation due to its density. Wahl observed the lateral angle to be sexually dimorphic in the 1980s. Previous studies showed various cut-off points to separate females from males, which are hardly replicable and difficult to apply. We want to test the Wahl method and compare it to a new landmark-based version. MATERIALS AND METHODS: In this study, we measured the lateral angle of 35 cremated petrous bones from late bronze age Austria using micro-CT scans. Technical errors of measurement were calculated for two different methods to intersect the internal acoustic meatus virtually in the midline (manual or landmark-based intersection). Furthermore, sex was estimated based on morphological features and metric measurements. This information was used in logistic regression modeling to define a cut-off point in our sample. RESULTS: The technical errors of measurement suggested that a landmark-based method was more precise in comparison to a manual intersection which was much more intuitive. Inter- and intra-observer errors were low which improved reliability. The logistic regression model produced good results in our sample (p = 0.02, R2 = 0.38, accuracy = 0.8). The mean lateral angle was similar to studies which focused on prehistoric cremated petrous bones. DISCUSSION: The proposed landmark-based method was precise, quick, and could be easily applied, even by unexperienced researchers. The size of the lateral angle seemed to be population-specific but also dependent on the method applied. We recommend to use the proposed landmark-based method which is more precise.

The effect of osteocyte-derived RANKL on bone graft remodeling: An in vivo experimental study.

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.

Functionalizing Collagen Membranes with MSC-Conditioned Media Promotes Guided Bone Regeneration in Rat Calvarial Defects.

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.

Impact of a Static Magnetic Field on Early Osseointegration: A Pilot Study in Canines.

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.

FasL is a catabolic factor in alveolar bone homeostasis.

AIM: Fas ligand (FasL) belongs to the tumour necrosis factor superfamily regulating bone turnover, inflammation, and apoptosis. The appendicular and axial skeleton phenotype of mature Faslgld mice has been reported. The impact of FasL on the alveolar bone providing support for the teeth at mature stages under healthy and induced inflammatory conditions remains unknown. MATERIALS AND METHODS: We performed a phenotypical analysis of mice carrying the homozygous Faslgld mutation and wild-type (WT) mice (C57BL/6) under healthy conditions and upon ligature-induced periodontitis. After 12 days, micro-computed tomography analysis revealed the distance between the cement enamel junction and the alveolar bone crest. Additional structural parameters, such as the bone volume fraction (BV/TV) and the periodontal ligament space volume, were measured. Histological analyses were performed to visualize the catabolic changes at the defect site. RESULTS: Healthy Faslgld mice were found to have more periodontal bone than their WT littermates. Faslgld had no significant effect on inflammatory osteolysis compared to WT controls with ligatures. Histology revealed eroded surfaces at the root and in the inter-proximal bone in both strains. CONCLUSIONS: Our findings suggest that FasL is a catabolic factor in alveolar bone homeostasis but it does not affect the inflammatory osteolysis.

Effects of Endochondral and Intramembranous Ossification Pathways on Bone Tissue Formation and Vascularization in Human Tissue-Engineered Grafts.

Bone grafts can be engineered by differentiating human mesenchymal stromal cells (MSCs) via the endochondral and intramembranous ossification pathways. We evaluated the effects of each pathway on the properties of engineered bone grafts and their capacity to drive bone regeneration. Bone-marrow-derived MSCs were differentiated on silk scaffolds into either hypertrophic chondrocytes (hyper) or osteoblasts (osteo) over 5 weeks of in vitro cultivation, and were implanted subcutaneously for 12 weeks. The pathways' constructs were evaluated over time with respect to gene expression, composition, histomorphology, microstructure, vascularization and biomechanics. Hypertrophic chondrocytes expressed higher levels of osteogenic genes and deposited significantly more bone mineral and proteins than the osteoblasts. Before implantation, the mineral in the hyper group was less mature than that in the osteo group. Following 12 weeks of implantation, the hyper group had increased mineral density but a similar overall mineral composition compared with the osteo group. The hyper group also displayed significantly more blood vessel infiltration than the osteo group. Both groups contained M2 macrophages, indicating bone regeneration. These data suggest that, similar to the body's repair processes, endochondral pathway might be more advantageous when regenerating large defects, whereas intramembranous ossification could be utilized to guide the tissue formation pattern with a scaffold architecture.

Bone Graft Packing and Its Association with Bone Regeneration in Maxillary Sinus Floor Augmentations: Histomorphometric Analysis of Human Biopsies.

Research in maxillary sinus floor augmentation (MSFA) focussed on the optimisation of microstructural parameters such as microporosity and particle size of bone substitute particles (BS). However, little is known about the impact of BS packing and the corresponding (void) interparticular space on bone regeneration. The aim of this study was to characterise the spatial distribution of BS and its association with BS integration 6 ± 1 months after MSFA. Histological thin-ground sections of 70 human sinus biopsies were histomorphometrically analysed: In serial zones of 100 µm proceeding from the sinus floor (SF) up to the apical end of the biopsy, we measured the distribution of BS particles within these zones in terms of volume (BSV/TV), number and size of BS particles, interparticle spacing (BS.Sp) and bone-to-BS contact. BS particles were not homogeneously distributed over the length of biopsies: The first 200 µm directly adjacent to the SF represented a zone poor in BS particles but with high osteogenic potential. Graft packing density increased from the SF towards the apical part of the AA. Integration of BS particles was inversely associated with the distance to the SF and the graft packing density. A high packing density through excessive compaction of BS particles should be avoided to optimise the macrostructural environment for bone regeneration.

Improved biomechanics in experimental chronic rotator cuff repair after shockwaves is not reflected by bone microarchitecture.

PURPOSE: The aim of this study was to investigate the effect of extracorporeal shockwave therapy (ESWT) on bone microstructure as well as the bone-tendon-interface and the musculo-tendinous transition zone to explain the previously shown improved biomechanics in a degenerative rotator cuff tear animal model. This study hypothesized that biomechanical improvements related to ESWT are a result of improved bone microstructure and muscle tendon properties. METHODS: In this controlled laboratory study unilateral supraspinatus (SSP) tendon detachment was performed in 48 male Sprague-Dawley rats. After a degeneration period of three weeks, SSP tendon was reconstructed transosseously. Rats were randomly assigned into three groups (n = 16 per group): control (noSW); intraoperative shockwave treatment (IntraSW); intra- and postoperative shockwave treatment (IntraPostSW). Eight weeks after SSP repair, all rats were sacrificed and underwent bone microstructure analysis as well as histological and immunohistochemical analyses. RESULTS: With exception of cortical porosity at the tendon area, bone microstructure analyses revealed no significant differences between the three study groups regarding cortical and trabecular bone parameters. Cortical Porosity at the Tendon Area was lowest in the IntraPostSW (p≤0.05) group. Histological analyses showed well-regenerated muscle and tendon structures in all groups. Immunohistochemistry detected augmented angiogenesis at the musculo-tendinous transition zone in both shockwave groups indicated by CD31 positive stained blood vessels. CONCLUSION: In conclusion, bone microarchitecture changes are not responsible for previously described improved biomechanical results after shockwave treatment in rotator cuff repair in rodents. Immunohistochemical analysis showed neovascularization at the musculo-tendinous transition zone within ESWT-treated animals. Further studies focusing on neovascularization at the musculo-tendinous transition zone are necessary to explain the enhanced biomechanical and functional properties observed previously. CLINICAL RELEVANCE: In patients treated with a double-row SSP tendon repair, an improvement in healing through ESWT, especially in this area, could prevent a failure of the medial row, which is considered a constantly observed tear pattern.

Ectopic Bone Tissue Engineering in Mice Using Human Gingiva or Bone Marrow-Derived Stromal/Progenitor Cells in Scaffold-Hydrogel Constructs.

Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation and bone regeneration capacity of mesenchymal stromal cells (MSC). Gingiva-derived progenitor cells (GPC) represent a less invasive alternative to bone marrow MSC (BMSC) for clinical applications. The aim of this study was to test the in vivo bone forming potential of human GPC and BMSC cultured as 3D spheroids or dissociated cells (2D). 2D and 3D cells encapsulated in constructs of human platelet lysate hydrogels (HPLG) and 3D-printed poly (L-lactide-co-trimethylene carbonate) scaffolds (HPLG-PLATMC) were implanted subcutaneously in nude mice; cell-free HPLG-PLATMC constructs served as a control. Mineralization was assessed using micro-computed tomography (µCT), histology, scanning electron microscopy (SEM) and in situ hybridization (ISH). After 4-8 weeks, µCT revealed greater mineralization in 3D-BMSC vs. 2D-BMSC and 3D-GPC (p < 0.05), and a similar trend in 2D-GPC vs. 2D-BMSC (p > 0.05). After 8 weeks, greater mineralization was observed in cell-free constructs vs. all 2D- and 3D-cell groups (p < 0.05). Histology and SEM revealed an irregular but similar mineralization pattern in all groups. ISH revealed similar numbers of 2D and 3D BMSC/GPC within and/or surrounding the mineralized areas. In summary, spheroid culture promoted ectopic mineralization in constructs of BMSC, while constructs of dissociated GPC and BMSC performed similarly. The combination of HPLG and PLATMC represents a promising scaffold for bone tissue engineering applications.

Bone regeneration in rat calvarial defects using dissociated or spheroid mesenchymal stromal cells in scaffold-hydrogel constructs.

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.

Osteoconductive Properties of a Volume-Stable Collagen Matrix in Rat Calvaria Defects: A Pilot Study.

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.

Osteoconductive properties of upside-down bilayer collagen membranes in rat calvarial defects.

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.

FasL Is Required for Osseous Healing in Extraction Sockets in Mice.

Fas ligand (FasL) is a member of the tumor necrosis factor (TNF) superfamily involved in the activation of apoptosis. Assuming that apoptosis is initiated after tooth extraction it is reasonable to suggest that FasL may play a pivotal role in the healing of extraction sockets. Herein, we tested the hypothesis of whether the lack of FasL impairs the healing of extraction sockets. To this end, we extracted upper right incisors of FasL knockout (KO) mice and their wildtype (WT) littermates. After a healing period of two weeks, bone volume over total volume (BV/TV) via µCT and descriptive histological analyses were performed. µCT revealed that BV/TV in the coronal region of the socket amounted to 39.4% in WT and 21.8% in KO, with a significant difference between the groups (p=0.002). Likewise, in the middle region of the socket, BV/TV amounted to 50.3% in WT and 40.8% in KO (p<0.001). In the apical part, however, no difference was noticed. Consistently, WT mice displayed a significantly higher median trabecular thickness and a lower trabecular separation when compared to the KO group at the coronal and central region of the socket. There was the overall tendency that in both, female and male mice, FasL affects bone regeneration. Taken together, these findings suggest that FasL deficiency may reduce bone regeneration during the healing process of extraction sockets.

Corrigendum: Ectopic Bone Tissue Engineering in Mice Using Human Gingiva or Bone Marrow-Derived Stromal/Progenitor Cells in Scaffold-Hydrogel Constructs.

[This corrects the article DOI: 10.3389/fbioe.2021.783468.].

Acid bone lysates reduce bone regeneration in rat calvaria defects.

Acid bone lysates (ABLs) represent the growth factors and other molecules released during autologous graft resorption. However, the impact of these bone-derived growth factors on the healing of bone defects has not yet been investigated. The aim of the present study was, therefore, to examine the impact of ABLs adsorbed to collagen membranes on bone regeneration. To this end, in 16 female Sprague Dawley rats, a standardized 5-mm-diameter critical size defect on the calvarial bone was created. The defects were covered with collagen membranes that had been soaked either in serum-free media or ABLs followed by lyophilization. After a healing period of 4 weeks, micro-computed tomography (µCT) and histological analyses by means of undecalcified thin ground sections were performed. µCT analysis of the inner 4 mm of the calvaria defect showed a greater bone defect coverage in the control group when compared to ABL group, 29.8% (confidence interval [CI]: 17.7-50.3) versus 5.6% (CI: 1.0-29.8, p = .03), respectively. Moreover, we found significantly more absolute bone volume (BV) in the control group when compared to ABL group, 0.59 mm3 (CI: 0.27-1.25) versus 0.07 mm3 (CI: 0.06-0.59, p = .04), respectively. Histomorphometry confirmed these findings with a relative BV in the central compartment of 14.1% (CI: 8.4-20.6) versus 5.6% (CI: 3.4-7.9, p = .004), respectively. These findings indicate that bone-derived growth factors contained in ABLs are able to attenuate bone regeneration within collagen membranes.

The vertical course of bone regeneration in maxillary sinus floor augmentations: A histomorphometric analysis of human biopsies.

BACKGROUND: Maxillary sinus floor augmentation (MSFA) is a well-established and predictable augmentation method in severely resorbed maxillae. However, data on the vertical course of bone graft consolidation within the maxillary sinus are rare. The aim of the present study was to quantify the vertical distribution of new bone formation (nBF) in MSFA and to characterize the vertical gradient of bone graft consolidation. METHODS: Eighty-five human sinus biopsies were harvested 6 ± 1 months after MSFA. Histological thin-ground sections were prepared and histomorphometrically analyzed. The volume of newly formed bone (nBV/TV) was measured in serial zones of 100 µm proceeding from the bottom of the sinus floor (SF) up to the apical top of the biopsy. The gradient of nBV/TV within the augmentation area was determined by the vertical distribution of nBV/TV along these zones. RESULTS: In the premolar region, nBV/TV slightly declined from 20.4% in the zone adjacent to the SF to 17.7% at a distance of 8 mm. The gradient was steeper in the molar region: nBV/TV decreased from 18.7% to 12.8%. This decline was even more distinct when the volume fraction and the height of the residual bone of the SF were low. CONCLUSIONS: nBF follows a gradient from native bone of the SF towards the apical part of the augmentation area. The distance to primordial bone thus plays a critical role for bone regeneration in MSFA, particularly in the molar region.