In Vivo Analysis of the Regeneration Capacity and Immune Response to Xenogeneic and Synthetic Bone Substitute Materials

Although various studies have investigated differences in the tissue reaction pattern to synthetic and xenogeneic bone substitute materials (BSMs), a lack of knowledge exists regarding the classification of both materials based on the DIN ISO 10993-6 scoring system, as well as the histomorphometrical measurement of macrophage subtypes within their implantation beds. Thus, the present study was conducted to analyze in vivo responses to both xenogeneic and synthetic bone substitute granules. A standardized calvaria implantation model in Wistar rats, in combination with established scoring, histological, histopathological, and histomorphometrical methods, was conducted to analyze the influence of both biomaterials on bone regeneration and the immune response. The results showed that the application of the synthetic BSM maxresorb^® induced a higher pro-inflammatory tissue response, while the xenogeneic BSM cerabone^® induced a higher anti-inflammatory reaction. Additionally, comparable bone regeneration amounts were found in both study groups. Histopathological scoring revealed that the synthetic BSM exhibited non-irritant scores at all timepoints using the xenogeneic BSM as control. Overall, the results demonstrated the biocompatibility of synthetic BSM maxresorb^® and support the conclusion that this material class is a suitable alternative to natural BSM, such as the analyzed xenogeneic material cerabone^®, for a broad range of indications.     

Ex Vivo and In Vivo Analyses of Novel 3D-Printed Bone Substitute Scaffolds Incorporating Biphasic Calcium Phosphate Granules for Bone Regeneration

(1) Background: The aim of this study was examining the ex vivo and in vivo properties of a composite made from polycaprolactone (PCL) and biphasic calcium phosphate (BCP) (synprint, ScientiFY GmbH) fabricated via fused deposition modelling (FDM); (2) Methods: Scaffolds were tested ex vivo for their mechanical properties using porous and solid designs. Subcutaneous implantation model analyzed the biocompatibility of PCL + BCP and PCL scaffolds. Calvaria implantation model analyzed the osteoconductive properties of PCL and PCL + BCP scaffolds compared to BCP as control group. Established histological, histopathological and histomorphometrical methods were performed to evaluate new bone formation.; (3) Results Mechanical testing demonstrated no significant differences between PCL and PCL + BCP for both designs. Similar biocompatibility was observed subcutaneously for PCL and PCL + BCP scaffolds. In the calvaria model, new bone formation was observed for all groups with largest new bone formation in the BCP group, followed by the PCL + BCP group, and the PCL group. This finding was influenced by the initial volume of biomaterial implanted and remaining volume after 90 days. All materials showed osteoconductive properties and PCL + BCP tailored the tissue responses towards higher cellular biodegradability. Moreover, this material combination led to a reduced swelling in PCL + BCP; (4) Conclusions: Altogether, the results show that the newly developed composite is biocompatible and leads to successful osteoconductive bone regeneration. The new biomaterial combines the structural stability provided by PCL with bioactive characteristics of BCP-based BSM. 3D-printed BSM provides an integration behavior in accordance with the concept of guided bone regeneration (GBR) by directing new bone growth for proper function and restoration.     

Effect of Different Bone Grafting Materials and Mesenchymal Stem Cells on Bone Regeneration: A Micro-Computed Tomography and Histomorphometric Study in a Rabbit Calvarial Defect Model

This study evaluated the new bone formation potential of micro–macro biphasic calcium phosphate (MBCP) and Bio-Oss grafting materials with and without dental pulp-derived mesenchymal stem cells (DPSCs) and bone marrow-derived mesenchymal stem cells (BMSCs) in a rabbit calvarial bone defect model. The surface structure of the grafting materials was evaluated using a scanning electron microscope (SEM). The multipotent differentiation characteristics of the DPSCs and BMSCs were assessed. Four circular bone defects were created in the calvarium of 24 rabbits and randomly allocated to eight experimental groups: empty control, MBCP, MBCP+DPSCs, MBCP+BMSCs, Bio-Oss+DPSCs, Bio-Oss+BMSCs, and autogenous bone. A three-dimensional analysis of the new bone formation was performed using micro-computed tomography (micro-CT) and a histological study after 2, 4, and 8 weeks of healing. Homogenously porous structures were observed in both grafting materials. The BMSCs revealed higher osteogenic differentiation capacities, whereas the DPSCs exhibited higher colony-forming units. The micro-CT and histological analysis findings for the new bone formation were consistent. In general, the empty control showed the lowest bone regeneration capacity throughout the experimental period. By contrast, the percentage of new bone formation was the highest in the autogenous bone group after 2 (39.4% ± 4.7%) and 4 weeks (49.7% ± 1.5%) of healing (p < 0.05). MBCP and Bio-Oss could provide osteoconductive support and prevent the collapse of the defect space for new bone formation. In addition, more osteoblastic cells lining the surface of the newly formed bone and bone grafting materials were observed after incorporating the DPSCs and BMSCs. After 8 weeks of healing, the autogenous bone group (54.9% ± 6.1%) showed a higher percentage of new bone formation than the empty control (35.3% ± 0.5%), MBCP (38.3% ± 6.0%), MBCP+DPSC (39.8% ± 5.7%), Bio-Oss (41.3% ± 3.5%), and Bio-Oss+DPSC (42.1% ± 2.7%) groups. Nevertheless, the percentage of new bone formation did not significantly differ between the MBCP+BMSC (47.2% ± 8.3%) and Bio-Oss+BMSC (51.2% ± 9.9%) groups and the autogenous bone group. Our study results demonstrated that autogenous bone is the gold standard. Both the DPSCs and BMSCs enhanced the osteoconductive capacities of MBCP and Bio-Oss. In addition, the efficiency of the BMSCs combined with MBCP and Bio-Oss was comparable to that of the autogenous bone after 8 weeks of healing. These findings provide effective strategies for the improvement of biomaterials and MSC-based bone tissue regeneration.     

Possible Role of Circulating Bone Marrow Mesenchymal Progenitors in Modulating Inflammation and Promoting Wound Repair

Circulating bone marrow mesenchymal progenitors (BMMPs) are known to be potent antigen-presenting cells that migrate to damaged tissue to secrete cytokines and growth factors. An altered or dysregulated inflammatory cascade leads to a poor healing outcome. A skin model developed in our previous study was used to observe the immuno-modulatory properties of circulating BMMP cells in inflammatory chronic wounds in a scenario of low skin perfusion. BMMPs were analysed exclusively and in conjunction with recombinant tumour necrosis factor alpha (TNFα) and recombinant hepatocyte growth factor (HGF) supplementation. We analysed the expression levels of interleukin-8 (IL-8) and ecto-5′-nucleotidase (CD73), together with protein levels for IL-8, stem cell factor (SCF), and fibroblast growth factor 1 (FGF-1). The successfully isolated BMMPs were positive for both hemopoietic and mesenchymal markers and showed the ability to differentiate into adipocytes, chondrocytes, and osteocytes. Significant differences were found in IL-8 and CD73 expressions and IL-8 and SCF concentrations, for all conditions studied over the three time points taken into consideration. Our data suggests that BMMPs may modulate the inflammatory response by regulating IL-8 and CD73 and influencing IL-8 and SCF protein secretions. In conclusion, we suggest that BMMPs play a role in wound repair and that their induced application might be suitable for scenarios with a low skin perfusion.     

Effect of Freshly Isolated Bone Marrow Mononuclear Cells and Cultured Bone Marrow Stromal Cells in Graft Cell Repopulation and Tendon-Bone Healing after Allograft Anterior Cruciate Ligament Reconstruction

Graft cell repopulation and tendon-bone tunnel healing are important after allograft anterior cruciate ligament reconstruction (ACLR). Freshly isolated bone marrow mononuclear cells (BMMNCs) have the advantage of short isolation time during surgery and may enhance tissue regeneration. Thus, we hypothesized that the effect of intra-articular BMMNCs in post-allograft ACLR treatment is comparable to that of cultured bone marrow stromal cells (BMSCs). A rabbit model of hamstring allograft ACLR was used in this study. Animals were randomly assigned to the BMMNC, BMSC, and control groups. Fresh BMMNCs isolated from the iliac crest during surgery and cultured BMSCs at passage four were used in this study. A total of 1 × 10⁷ BMMNCs or BMSCs in 100 µL phosphate-buffered saline were injected into the knee joint immediately after ACLR. The control group was not injected with cells. At two and six weeks post operation, we assessed graft cell repopulation with histological and cell tracking staining (PKH26), and tendon-bone healing with histological micro-computed tomography and immunohistochemical analyses for collagen I and monocyte chemoattractant protein-1 (MCP1). At two weeks post operation, there was no significant difference in the total cell population within the allograft among the three groups. However, the control group showed significantly higher cell population within the allograft than that of BM cell groups at six weeks. Histological examination of proximal tibia revealed that the intra-articular delivered cells infiltrated into the tendon-bone interface. Compared to the control group, the BM cell groups showed broader gaps with interfacial fibrocartilage healing, similar collagen I level, and higher MCP1 expression in the early stage. Micro-CT did not reveal any significant difference among the three groups. BMMNCs and BMSCs had comparable effects on cell repopulation and interfacial allograft-bone healing. Intra-articular BM cells delivery had limited benefits on graft cell repopulation and caused higher inflammation than that in the control group in the early stage, with fibrocartilage formation in the tendon-bone interface after allograft ACLR.     

BMP3 Affects Cortical and Trabecular Long Bone Development in Mice

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3^−/− mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3^−/− mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3^−/− mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3^−/− mice.     

Early Bone Healing on Hydroxyapatite-Coated and Chemically-Modified Hydrophilic Implant Surfaces in an Ovine Model

Implant topography affects early peri-implant bone healing by changing the osteoconduction rate in the surrounding biological environment. Implant surfaces have been designed to promote faster and stronger bone formation for rapid and stable prosthesis loading. Early peri-implant bone healing has been observed with a sandblasted, acid-etched implant that was chemically modified to be hydrophilic (cmSLA). The present study investigates whether early peri-implant bone healing extends to a rough surface implant with a high crystalline hydroxyapatite surface (TSV MP-1 HA). Three implants were randomly placed in porous trabecular bone within both medial femoral condyles of 10 sheep. Early peri-implant bone stability was measured at 3- and 6-weeks healing time following implant insertion. Results indicated a similar implant stability quotient between the implants at insertion and over time. The significant increase over time of reverse torque values with respect to insertion torque (p < 0.001) did not differ between the implants. However, the bone-to-implant contact of TSV MP-1 HA was significantly higher than that of cmSLA implants at 6 weeks (p < 0.01). These data validate previous findings of a hydrophilic implant surface and extend the observation of early osseointegration to a rough surface implant in porous trabecular bone.     

Stimulation of Bone Healing by Sustained Bone Morphogenetic Protein 2 (BMP-2) Delivery

The aim of the study was to investigate the effect of a sustained release of bone morphogenetic protein2 (BMP-2) incorporated in a polymeric implant coating on bone healing. In vitro analysis revealed a sustained, but incomplete BMP-2 release until Day 42. For the in vivo study, the rat tibia osteotomy was stabilized either with control or BMP-2 coated wires, and the healing progress was followed by micro computed tomography (μCT), biomechanical testing and histology at Days 10, 28, 42 and 84. MicroCT showed an accelerated formation of mineralized callus, as well as remodeling and an increase of mineralized/total callus volume (p = 0.021) at Day 42 in the BMP-2 group compared to the control. Histology revealed an increased callus mineralization at Days 42 and 84 (p = 0.006) with reduced cartilage at Day 84 (p = 0.004) in the BMP-2 group. Biomechanical stiffness was significantly higher in the BMP-2 group (p = 0.045) at Day 42. In summary, bone healing was enhanced after sustained BMP-2 application compared to the control. Using the same drug delivery system, but a burst release of BMP-2, a previous published study showed a similar positive effect on bone healing. Distinct differences in the healing outcome might be explained due to the different BMP release kinetics and dosages. However, further studies are necessary to adapt the optimal release profiles to physiological mechanisms.     

Bone Tissue Engineering in Rat Calvarial Defects Using Induced Bone-like Tissue by rhBMPs from Immature Muscular Tissues In Vitro

This study aimed to induce bone-like tissue from immature muscular tissue (IMT) in vitro using commercially available recombinant human bone morphogenetic protein (rhBMP)-2, rhBMP-4, and rhBMP-7, and then implanting this tissue into a calvarial defect in rats to assess healing. IMTs were extracted from 20-day-old Sprague-Dawley (SD) fetal rats, placed on expanded polytetrafluoroethylene (ePTFE) with 10 ng/μL each of rhBMP-2, BMP-4, and BMP-7, and cultured for two weeks. The specimens were implanted into calvarial defects in 3-week-old SD rats for up to three weeks. Relatively strong radiopacity was observed on micro-CT two weeks after culture, and bone-like tissue, comprising osteoblastic cells and osteoids, was partially observed by H&E staining. Calcium, phosphorus, and oxygen were detected in the extracellular matrix using an electron probe micro analyzer, and X-ray diffraction patterns and Fourier transform infrared spectroscopy spectra of the specimen were found to have typical apatite crystal peaks and spectra, respectively. Furthermore, partial strong radiopacity and ossification were confirmed one week after implantation, and a dominant novel bone was observed after two weeks in the defect site. Thus, rhBMP-2, BMP-4, and BMP-7 differentiated IMT into bone-like tissue in vitro, and this induced bone-like tissue has ossification potential and promotes the healing of calvarial defects. Our results suggest that IMT is an effective tissue source for bone tissue engineering.     

Facilitation of Bone Healing Processes Based on the Developmental Function of Meox2 in Tooth Loss Lesion

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson’s trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including α-SMA, CK14, IL-1β, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.     

CSBD Healing in Rats after Application of Bovine Xenogeneic Biomaterial Enriched with Magnesium Alloy

Xenogeneic biomaterials Cerbone^® and OsteoBiol^® are widely used in oral implantology. In dental practice, xenogeneic biomaterial is usually combined with autologous bone to provide bone volume stability needed for long-term dental implants. Magnesium alloy implants dissolve and form mineral corrosion layer that is directly in contact with bone tissue, allowing deposition of the newly formed bone. CSBD heals by intramembranous ossification and therefore is a convenient model for analyses of ostoconductive and osteoinductive properties of different type of biomaterials. Magnesium alloy-enriched biomaterials have not yet been applied in oral implantology. Therefore, the aim of the current study was to investigate biological properties of potentially new bovine xenogeneic biomaterial enriched with magnesium alloy in a 5 mm CSBD model. Osteoconductive properties of Cerabone^®, Cerabone^® + Al. bone, and OsteoBiol^® were also analyzed. Dynamics of bone healing was followed up on the days 3, 7, 15, 21, and 30. Calvary bone samples were analyzed by micro-CT, and values of the bone morphometric parameters were assessed. Bone samples were further processed for histological and immunohistochemical analyses. Histological observation revealed CSBD closure at day 30 of the given xenogeneic biomaterial groups, with the exception of the control group. TNF-α showed high intensity of expression at the sites of MSC clusters that underwent ossification. Osx was expressed in pre-osteoblasts, which were differentiated into mature osteoblasts and osteocytes. Results of the micro-CT analyses showed linear increase in bone volume of all xenogeneic biomaterial groups and also in the control. The highest average values of bone volume were found for the Cerabone^® + Mg group. In addition, less residual biomaterial was estimated in the Cerabone^® + Mg group than in the Cerabone^® group, indicating its better biodegradation during CSBD healing. Overall, the magnesium alloy xenogeneic biomaterial demonstrated key properties of osteoinduction and biodegradidibility during CSBD healing, which is the reason why it should be recommended for application in clinical practice of oral implantology.     

Synthesis and Evaluation of a Chitosan–Silica-Based Bone Substitute for Tissue Engineering

Bone defects have prompted the development of biomaterial-based bone substitutes for restoring the affected tissue completely. Although many biomaterials have been designed and evaluated, the combination of properties required in a biomaterial for bone tissue engineering still poses a challenge. In this study, a chitosan–silica-based biocomposite was synthetized, and its physicochemical characteristics and biocompatibility were characterized, with the aim of exploring the advantages and drawbacks of its use in bone tissue engineering. Dynamic light scattering measurements showed that the mean hydrodynamic size of solid silica particles (Sol-Si) was 482 ± 3 nm. Scanning electron microscopy of the biocomposite showed that Sol-Si were homogenously distributed within the chitosan (CS) matrix. The biocomposite swelled rapidly and was observed to have no cytotoxic effect on the [3T3] cell line within 24 h. Biocompatibility was also analyzed in vivo 14 days post-implant using a murine experimental model (Wistar rats). The biocomposite was implanted in the medullary compartment of both tibiae (n = 12). Histologically, no acute inflammatory infiltrate or multinucleated giant cells associated to the biocomposite were observed, indicating good biocompatibility. At the tissue–biocomposite interface, there was new formation of woven bone tissue in close contact with the biocomposite surface (osseointegration). The new bone formation may be attributed to the action of silica. Free silica particles originating from the biocomposite were observed at the tissue–biocomposite interface. According to our results, the biocomposite may act as a template for cellular interactions and extracellular matrix formation, providing a structural support for new bone tissue formation. The CS/Sol-Si biocomposite may act as a Si reservoir, promoting new bone formation. A scaffold with these properties is essential for cell differentiation and filling a bone defect.     

重组人骨形态发生蛋白-7复合体对牙槽骨再生的影响

目的观察基因重组人骨形态发生蛋白-7(rhBMP-7)与明胶海绵的复合体对犬牙槽骨再生的影响。方法犬拔牙创内植入rhBMP-7与明胶海绵的复合体,以自然愈合拔牙创为对照。于术后2、4、8周分别将犬处死,取拔牙创处牙槽骨标本,行X线和组织学切片检查,观察成骨情况及牙槽骨萎缩情况。结果X线及组织学切片显示实验侧成骨速度及成骨质量明显优于对照侧。结论rhBMP-7与明胶海绵复合体是一种可促进牙槽骨再生的新型生物复合材料。     

In Vivo Efficacy of Neutrophil-Mediated Bone Regeneration Using a Rabbit Calvarial Defect Model

Reconstruction of bone due to surgical removal or disease-related bony defects is a clinical challenge. It is known that the immune system exerts positive immunomodulatory effects on tissue repair and regeneration. In this study, we evaluated the in vivo efficacy of autologous neutrophils on bone regeneration using a rabbit calvarial defect model. Methods: Twelve rabbits, each with two surgically created calvarial bone defects (10 mm diameter), were randomly divided into two groups; (i) single application of neutrophils (SA-NP) vs. SA-NP control, and (ii) repetitive application of neutrophils (RA-NP) vs. RA-NP control. The animals were euthanized at 4 and 8 weeks post-operatively and the treatment outcomes were evaluated by micro-computed tomography, histology, and histomorphometric analyses. Results: The micro-CT analysis showed a significantly higher bone volume fraction (bone volume/total volume) in the neutrophil-treated groups, i.e., median interquartile range (IQR) SA-NP (18) and RA-NP (24), compared with the untreated controls, i.e., SA-NP (7) and RA-NP (14) at 4 weeks (p < 0.05). Similarly, new bone area fraction (bone area/total area) was significantly higher in neutrophil-treated groups at 4 weeks (p < 0.05). Both SA-NP and RA-NP had a considerably higher bone volume and bone area at 8 weeks, although the difference was not statistically significant. In addition, immunohistochemical analysis at 8 weeks revealed a higher expression of osteocalcin in both SA-NP and RA-NP groups. Conclusions: The present study provides first hand evidence that autologous neutrophils may have a positive effect on promoting new bone formation. Future studies should be performed with a larger sample size in non-human primate models. If proven feasible, this new promising strategy could bring clinical benefits for bone defects to the field of oral and maxillofacial surgery.     

Systemic Dietary Hesperidin Modulation of Osteoclastogenesis,Bone Homeostasis and Periodontal Disease in Mice

This study aimed to evaluate the effects of hesperidin (HE) on in vitro osteoclastogenesis and dietary supplementation on mouse periodontal disease and femoral bone phenotype. RAW 264.7 cells were stimulated with RANKL in the presence or absence of HE (1, 100 or 500 µM) for 5 days, and evaluated by TRAP, TUNEL and Western Blot (WB) analyses. In vivo, C57BL/6 mice were given HE via oral gavage (125, 250 and 500 mg/kg) for 4 weeks. A sterile silk ligature was placed between the first and second right maxillary molars for 10 days and microcomputed tomography (μCT), histopathological and immunohistochemical evaluation were performed. Femoral bones subjected or not to dietary HE (500 mg/kg) for 6 and 12 weeks were evaluated using μCT. In vitro, HE 500 µM reduced formation of RANKL-stimulated TRAP-positive(+) multinucleated cells (500 µM) as well as c-Fos and NFATc1 protein expression (p < 0.05), markers of osteoclasts. In vivo, dietary HE 500 mg/kg increased the alveolar bone resorption in ligated teeth (p < 0.05) and resulted in a significant increase in TRAP+ cells (p < 0.05). Gingival inflammatory infiltrate was greater in the HE 500 mg/kg group even in the absence of ligature. In femurs, HE 500 mg/kg protected trabecular and cortical bone mass at 6 weeks of treatment. In conclusion, HE impaired in vitro osteoclastogenesis, but on the contrary, oral administration of a high concentration of dietary HE increased osteoclast numbers and promoted inflammation-induced alveolar bone loss. However, HE at 500 mg/kg can promote a bone-sparing effect on skeletal bone under physiological conditions.     

Antiosteoporotic Nanohydroxyapatite Zoledronate Scaffold Seeded with Bone Marrow Mesenchymal Stromal Cells for Bone Regeneration: A 3D In Vitro Model

Background: Bisphosphonates are widely employed drugs for the treatment of pathologies with high bone resorption, such as osteoporosis, and display a great affinity for calcium ions and apatitic substrates. Here, we aimed to investigate the potentiality of zoledronate functionalized hydroxyapatite nanocrystals (HAZOL) to promote bone regeneration by stimulating adhesion, viability, metabolic activity and osteogenic commitment of human bone marrow derived mesenchymal stromal cells (hMSCs). Methods: we adopted an advanced three-dimensional (3D) in vitro fracture healing model to study porous scaffolds: hMSCs were seeded onto the scaffolds that, after three days, were cut in halves and unseeded scaffolds were placed between the two halves. Scaffold characterization by X-ray diffraction, transmission and scanning electron microscopy analyses and cell morphology, viability, osteogenic differentiation and extracellular matrix deposition were evaluated after 3, 7 and 10 days of culture. Results: Electron microscopy showed a porous and interconnected structure and a uniform cell layer spread onto scaffolds. Scaffolds were able to support cell growth and cells progressively colonized the whole inserts in absence of cytotoxic effects. Osteogenic commitment and gene expression of hMSCs were enhanced with higher expressions of ALPL, COL1A1, BGLAP, RUNX2 and Osterix genes. Conclusion: Although some limitations affect the present study (e.g., the lack of longer experimental times, of mechanical stimulus or pathological microenvironment), the obtained results with the adopted experimental setup suggested that zoledronate functionalized scaffolds (GHAZOL) might sustain not only cell proliferation, but positively influence osteogenic differentiation and activity if employed in bone fracture healing.     

A Review of Recent Developments in the Molecular Mechanisms of Bone Healing

Between 5 and 10 percent of fractures do not heal, a condition known as nonunion. In clinical practice, stable fracture fixation associated with autologous iliac crest bone graft placement is the gold standard for treatment. However, some recalcitrant nonunions do not resolve satisfactorily with this technique. For these cases, biological alternatives are sought based on the molecular mechanisms of bone healing, whose most recent findings are reviewed in this article. The pro-osteogenic efficacy of morin (a pale yellow crystalline flavonoid pigment found in old fustic and osage orange trees) has recently been reported, and the combined use of bone morphogenetic protein-9 (BMP9) and leptin might improve fracture healing. Inhibition with methyl-piperidino-pyrazole of estrogen receptor alpha signaling delays bone regeneration. Smoking causes a chondrogenic disorder, aberrant activity of the skeleton’s stem and progenitor cells, and an intense initial inflammatory response. Smoking cessation 4 weeks before surgery is therefore highly recommended. The delay in fracture consolidation in diabetic animals is related to BMP6 deficiency (35 kDa). The combination of bioceramics and expanded autologous human mesenchymal stem cells from bone marrow is a new and encouraging alternative for treating recalcitrant nonunions.     

Myricetin Prevents Alveolar Bone Loss in an Experimental Ovariectomized Mouse Model of Periodontitis

Periodontitis is a common chronic inflammatory disease, which leads to alveolar bone resorption. Healthy and functional alveolar bone, which can support the teeth and enable their movement, is very important for orthodontic treatment. Myricetin inhibited osteoclastogenesis by suppressing the expression of some genes, signaling pathways, and cytokines. This study aimed to investigate the effects of myricetin on alveolar bone loss in an ovariectomized (OVX) mouse model of periodontitis as well as in vitro osteoclast formation and bone resorption. Twenty-four healthy eight-week-old C57BL/J6 female mice were assigned randomly to four groups: phosphate-buffered saline (PBS) control (sham) OVX + ligature + PBS (vehicle), and OVX + ligature + low or high (2 or 5 mg∙kg⁻¹∙day⁻¹, respectively) doses of myricetin. Myricetin or PBS was injected intraperitoneally (i.p.) every other day for 30 days. The maxillae were collected and subjected to further examination, including micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, and tartrate-resistant acid phosphatase (TRAP) staining; a resorption pit assay was also performed in vitro to evaluate the effects of myricetin on receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. Myricetin, at both high and low doses, prevented alveolar bone resorption and increased alveolar crest height in the mouse model and inhibited osteoclast formation and bone resorption in vitro. However, myricetin was more effective at high dose than at low dose. Our study demonstrated that myricetin had a positive effect on alveolar bone resorption in an OVX mouse model of periodontitis and, therefore, may be a potential agent for the treatment of periodontitis and osteoporosis.     

Bone Healing Materials in the Treatment of Recalcitrant Nonunions and Bone Defects

The usual treatment for bone defects and recalcitrant nonunions is an autogenous bone graft. However, due to the limitations in obtaining autogenous bone grafts and the morbidity associated with their procurement, various bone healing materials have been developed in recent years. The three main treatment strategies for bone defects and recalcitrant nonunions are synthetic bone graft substitutes (BGS), BGS combined with bioactive molecules, and BGS and stem cells (cell-based constructs). Regarding BGS, numerous biomaterials have been developed to prepare bone tissue engineering scaffolds, including biometals (titanium, iron, magnesium, zinc), bioceramics (hydroxyapatite (HA)), tricalcium phosphate (TCP), biopolymers (collagen, polylactic acid (PLA), polycaprolactone (PCL)), and biocomposites (HA/MONs@miR-34a composite coating, Bioglass (BG)-based ABVF-BG (antibiotic-releasing bone void filling) putty). Bone tissue engineering scaffolds are temporary implants that promote tissue ingrowth and new bone regeneration. They have been developed to improve bone healing through appropriate designs in terms of geometric, mechanical, and biological performance. Concerning BGS combined with bioactive molecules, one of the most potent osteoinductive growth factors is bone morphogenetic proteins (BMPs). In recent years, several natural (collagen, fibrin, chitosan, hyaluronic acid, gelatin, and alginate) and synthetic polymers (polylactic acid, polyglycolic acid, polylactic-coglycolide, poly(e-caprolactone) (PCL), poly-p-dioxanone, and copolymers consisting of glycolide/trimethylene carbonate) have been investigated as potential support materials for bone tissue engineering. Regarding BGS and stem cells (cell-based constructs), the main strategies are bone marrow stromal cells, adipose-derived mesenchymal cells, periosteum-derived stem cells, and 3D bioprinting of hydrogels and cells or bioactive molecules. Currently, significant research is being performed on the biological treatment of recalcitrant nonunions and bone defects, although its use is still far from being generalized. Further research is needed to investigate the efficacy of biological treatments to solve recalcitrant nonunions and bone defects.