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

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

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.publishedVersio

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

From Mouth to Model: Combining in vivo and in vitro oral biofilm growth

Oral biofilm studies based on simplified experimental setups are difficult to interpret. Models are limited mostly by the number of bacterial species observed and the insufficiency of artificial media. Few studies have attempted to overcome these limitations and to cultivate native oral biofilm. This study aimed to grow oral biofilm in vivo before transfer to a biofilm reactor for ex-situ incubation. The in-vitro survival of this oral biofilm and the changes in bacterial composition over time were observed. Six human enamel-dentin slabs embedded buccally in dental splints were used as biofilm carriers. Fitted individually to the upper jaw of 25 non-smoking male volunteers, the splints were worn continuously for 48 hours. During this time, tooth-brushing and alcohol-consumption were not permitted. The biofilm was then transferred on slabs into a biofilm reactor and incubated there for 48 hours while being nourished in BHI medium. Live/dead staining and confocal laser scanning microscopy were used to observe bacterial survival over four points in time: directly after removal (T0) and after 1h (T1), 24h (T2) and 48h (T3) of incubation. Bacterial diversity at T0 and T3 was compared with 454-pyrosequencing. Fluorescence in situ hybridization was performed to show specific taxa. Survival curves were calculated with a specially designed MATLAB script. Acacia and QIIME 1.9.1 were used to process pyrosequencing data. SPSS 21.0 and R 3.3.1 were used for statistical analysis.After initial fluctuations at T1, survival curves mostly showed approximation of the bacterial numbers to the initial level at T3. Pyrosequencing analysis resulted in 117 OTUs common to all samples. The genera Streptococcus and Veillonella (both Firmicutes) dominated at T0 and T3. They make up two thirds of the biofilm. Genera with lower relative abundance had grown significantly at T3. FISH analysis confirmed the pyrosequencing results, i.e. the predominant staining of Firmicutes. We demonstrate the in vitro survival of native primary oral biofilm in its natural complexity over 48 hours. Our results offer a baseline for cultivation studies of native oral biofilms in (phyto-) pharmacological and dental materials research. Further investigations and validation of culturing conditions could also facilitate the study of biofilm-induced diseases

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 mm$^{3}$ (CI: 0.27-1.25) versus 0.07 mm$^{3}$ (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

DBBM shows no signs of resorption under inflammatory conditions. An experimental study in the mouse calvaria.

OBJECTIVES Deproteinized bovine bone mineral (DBBM) is not resorbable. However, the behavior of DBBM under inflammatory conditions remains unclear. Aim of the study was therefore to evaluate the resorption of DBBM under local inflammatory conditions in vivo using the calvarial osteolysis model. METHODS In thirty adult BALB/c mice, DBBM was implanted into the space between the elevated soft tissue and the calvarial bone. Inflammation was induced either by lipopolysaccharides (LPS) injection or by polyethylene particles (Ceridust) mixed with DBBM. Three modalities were randomly applied (n = 10 each): (a) DBBM alone (control), (b) DBBM + LPS, and (c) DBBM + polyethylene particles (Ceridust). Mice were euthanized on day fourteen, and each calvarium was subjected to histological and µCT analysis. Primary outcome was the size distribution of the DBBM particles. Secondary outcome was the surface erosion of the calvarial bone. RESULTS Histological and µCT analysis revealed that the size distribution and the volume of DBBM particles in the augmented site were similar between DBBM alone and the combinations with LPS or polyethylene particles. Moreover, histological evaluation showed no signs of erosions of DBBM particles under inflammatory conditions. µCT analysis and histology further revealed that LPS and the polyethylene particles, but not the DBBM alone, caused severe erosions of the calvarial bone as indicated by large voids representing the massive compensatory new immature woven bone formation on the endosteal surface. CONCLUSIONS Local calvarial bone but not the DBBM particles undergo severe resorption and subsequent new bone formation under inflammatory conditions in a mouse model

The Effect of Parathyroid Hormone on Osseointegration in Insulin-Treated Diabetic Rats.

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 insulin-controlled 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