Rat Critical-sized Calvarial Defect Model Research Articles

CXCL12 overexpression promotes the angiogenesis potential of periodontal ligament stem cells

Periodontal ligament stem cells (PDLSCs) are a major source of mesenchymal stem cells (MSCs) in adults and are effective for tissue engineering, like promoting angiogenesis and bone regeneration. CXCL12 has been reported to be involved in the recruitment and engraftment of MSCs in wound sites. However, whether CXCL12 potentiates the angiogenesis of PDLSCs is not clear. In this experiment, we transduced PDLSCs with CXCL12, and evaluated the angiogenesis potential of CXCL12-modified PDLSCs through in vitro and in vivo studies. The results showed that CXCL12 overexpression significantly stimulated the gene and protein expressions of bFGF, VEGF, SCF and PLGF in PDLSCs; CXCL12 gene modified PDLSCs formed longer capillary‐like structure; Moreover, in vivo transplanted PDLSCs transduced with CXCL12 could significantly promote bone tissue repair and angiogenesis in a rat critical-sized calvarial bone defect model. Taken together, our study confirmed that CXCL12 can enhance the angiogenesis potential of PDLSCs, which are crucial in the repair and regeneration of bone tissue.

Local application of lactoferrin promotes bone regeneration in a rat critical-sized calvarial defect model as demonstrated by micro-CT and histological analysis.

Lactoferrin is a multifunctional glycoprotein with therapeutic potential for bone tissue engineering. The aim of this study was to assess the efficacy of local application of lactoferrin on bone regeneration. Five‐millimetre critical‐sized defects were created over the right parietal bone in 64 Sprague–Dawley rats. The rats were randomized into four groups: group 1 (n = 20) had empty defects; group 2 (n = 20) had defects grafted with collagen gels (3 mg/ml); group 3 (n = 20) had defects grafted with collagen gels impregnated with bovine lactoferrin (10 μg/gel); and group 4 (n = 4) had sham surgeries (skin and periosteal incisions only). The rats were sacrificed at 4 or 12 weeks post‐operatively, and the calvaria were excised and evaluated with micro‐CT (Skyscan 1172) followed by histology. The bone volume fraction (BV/TV) was higher in lactoferrin‐treated animals at both timepoints, with groups 1, 2, 3 and 4 measuring 10.5 ± 1.1%, 8.6 ± 1.4%, 16.5 ± 0.6% and 24.27 ± 2.6%, respectively, at 4 weeks (P < 0.05); and 12.2 ± 1.3%, 13.6 ± 1.5%, 21.9 ± 1.2% and 29.3 ± 0.8%, respectively, at 12 weeks (P < 0.05). Histological analysis revealed that the newly formed bone within the calvarial defects of all groups was a mixture of woven and lamellar bone, with more bone in the group treated with lactoferrin at both timepoints. Our study demonstrated that local application of lactoferrin significantly increased bone regeneration in a rat critical‐sized calvarial defect model. The profound effect of lactoferrin on bone regeneration has therapeutic potential to improve the poor clinical outcomes associated with bony non‐union. LF In Vivo JTERM Authors Contributions. Copyright © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.

Strontium-Doped Amorphous Calcium Phosphate Porous Microspheres Synthesized through a Microwave-Hydrothermal Method Using Fructose 1,6-Bisphosphate as an Organic Phosphorus Source: Application in Drug Delivery and Enhanced Bone Regeneration.

Nanostructured calcium phosphate porous microspheres are of great potential in drug delivery and bone regeneration due to their large specific surface area, biocompatibility, and similarity to inorganic component of osseous tissue. In this work, strontium (Sr)-doped amorphous calcium phosphate porous microspheres (SrAPMs) were synthesized through a microwave-hydrothermal method using fructose 1,6-bisphosphate trisodium salt as the source of phosphate ions. The SrAPMs showed a mesoporous structure and a relatively high specific area. Compared with the hydroxyapatite nanorods prepared by using Na2HPO4·12H2O as the phosphorus source, the SrAPMs with a higher specific surface area were more effective in drug loading using vancomycin as the antiobiotics of choice and consequently having a higher antibacterial efficiency both on agar plates and in broths. Furthermore, to assess the potential application of SrAPMs in bone defect repair, a novel biomimetic bone tissue-engineering scaffold consisting of collagen (Coll) and SrAPMs was constructed using a freeze-drying fabrication process. Incorporation of the SrAPMs not only improved the mechanical properties, but also enhanced the osteogenesis of rat bone marrow mesenchymal stem cells. The in vivo experiments demonstrated that the SrAPMs/Coll scaffolds remarkably enhanced new bone formation compared with the Coll and APMs/Coll scaffolds in a rat critical-sized calvarial defect model at 8 weeks postimplantation. In summary, SrAPMs developed in this work are promising as antibiotic carriers and may encourage bone formation when combined with collagen.

Improving osteogenesis of three-dimensional porous scaffold based on mineralized recombinant human-like collagen via mussel-inspired polydopamine and effective immobilization of BMP-2-derived peptide

An ideal bone substitute should be biocompatible, biodegradable, osteoinductive and osteoconductive. In our previous work, we fabricated a three-dimensional porous scaffold based on mineralized recombinant human-like collagen, nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA). Like other HA/collagen scaffolds, the nHA/RHLC/PLA scaffold lacked osteoinductive bioactivity. The purpose of the present study was to develop a polydopamine (pDA)-assisted BMP-2-derived peptide (designated as P24) surface modification strategy for improving the osteogenesis of the nHA/RHLC/PLA scaffold. The immobilization efficiency and release kinetics of P24, and in vitro osteoinductive activity of the nHA/RHLC/PLA-pDA-P24 scaffold were examined. The in vivo osteoinductive activity of the scaffold was evaluated usinga rat criticalsize calvarial defect model. Our results showed that pDA-assisted surface modification could more efficiently mediate the immobilization of P24 peptide onto the scaffold surfaces than physical adsorption. The in vitro release study showed that the P24 peptide was released slowly and steadily from the nHA/RHLC/PLA-pDA-P24 scaffold in a sustained manner, with a short initial burst release only during the first day, while the physisorbed nHA/RHLC/PLA-P24 group showed a sharp burst P24 release followed by a plateau phase. In vitro osteogenesis assay, the ALP activitiy and mRNA expression of osteo-specific markers of rat-derived mesenchymal stem cells (rMSCs) in the nHA/RHLC/PLA-pDA-P24 group were significantly higher than those of the nHA/RHLC/PLA-P24 and non-P24-loaded nHA/RHLC/PLA groups. In vivo, three-dimensional CT evaluation and histological examination demonstrated the nHA/RHLC/PLA-pDA-P24 scaffolds significantly enhanced bone regeneration of rat cranial defects to a much greater extent than physisorbed nHA/RHLC/PLA-P24 and non-P24-loaded nHA/RHLC/PLA scaffolds. Our findings indicated that the pDA-assisted surface modification method could significantly improve the osteogenesis activity of the nHA/RHLC/PLA scaffold and the new nHA/RHLC/PLA-pDA-P24 scaffold was a promising scaffold biomaterial for bone tissue regeneration.

Accelerated craniofacial bone regeneration through dense collagen gel scaffolds seeded with dental pulp stem cells.

Therapies using mesenchymal stem cell (MSC) seeded scaffolds may be applicable to various fields of regenerative medicine, including craniomaxillofacial surgery. Plastic compression of collagen scaffolds seeded with MSC has been shown to enhance the osteogenic differentiation of MSC as it increases the collagen fibrillary density. The aim of the present study was to evaluate the osteogenic effects of dense collagen gel scaffolds seeded with mesenchymal dental pulp stem cells (DPSC) on bone regeneration in a rat critical-size calvarial defect model. Two symmetrical full-thickness defects were created (5 mm diameter) and filled with either a rat DPSC-containing dense collagen gel scaffold (n = 15), or an acellular scaffold (n = 15). Animals were imaged in vivo by microcomputer tomography (Micro-CT) once a week during 5 weeks, whereas some animals were sacrificed each week for histology and histomorphometry analysis. Bone mineral density and bone micro-architectural parameters were significantly increased when DPSC-seeded scaffolds were used. Histological and histomorphometrical data also revealed significant increases in fibrous connective and mineralized tissue volume when DPSC-seeded scaffolds were used, associated with expression of type I collagen, osteoblast-associated alkaline phosphatase and osteoclastic-related tartrate-resistant acid phosphatase. Results demonstrate the potential of DPSC-loaded-dense collagen gel scaffolds to benefit of bone healing process.

Systemic and local zoledronic acid treatment with hydroxyapatite bone graft: A histological and histomorphometric experimental study.

In this study, the aim was to compare the relative efficacy of systemic and local zoledronic acid (ZA) on a hydroxyapatite (HA) bone graft in a rat critical-size calvarial bone defect. In total, 84 female rats were divided into four groups: Empty control (EC) group with no treatment applied; HA group, in which only HA bone graft material was used in the calvarium; and HA plus local ZA (HA+LZA) and HA plus systemic ZA (HA+SZA) groups, in which animals received ZA locally or systemically, respectively, with HA bone graft material in the calvarium. A 5-mm standardised critical-size calvarial bone defect was created with a standard trephine drill and the respective treatment was applied. Rats were sacrificed 7, 14 and 28 days later. The numbers of osteoclasts and osteoblasts, and degree of bone formation were evaluated histopathologically and histomorphometrically. Statistically significant differences were detected between the HA, HA+LZA and HA+SZA groups and the EC group for new bone formation (P<0.05). Osteoblast numbers in the HA+LZA and HA+SZA groups were significantly higher compared with those in the EC and HA groups (P<0.05). No statistically significant difference was detected between the HA+LZA and HA+SZA groups in new bone formation or osteoblast number (P>0.05). Bone formation was significantly higher in the HA group than in the EC group (P<0.05). The numbers of osteoclasts in the HA+LZA and HA+SZA groups were significantly higher than those in the groups EC and HA (P<0.05); however, there was no significant difference between groups HA+LZA and HA+SZA (P>0.05). Within the limitations of this study, systemic or local administration of ZA enhanced new bone formation with a HA bone graft in a rat critical-size calvarial defect model.

Amorphous Silica: A New Antioxidant Role for Rapid Critical-Sized Bone Defect Healing.

Traumatic fractures cause structurally unstable sites due to severe bone loss. Such fractures generate a high yield of reactive oxygen species (ROS) that can lead to oxidative stress. Excessive and prolonged ROS activity impedes osteoblast differentiation and instigates long healing times. Stimulation of antioxidants such as superoxide dismutase (SOD1), are crucial to reduce ROS, stimulate osteogenesis, and strengthen collagen and mineral formation. Yet, no current fixative devices have shown an ability to enhance collagen matrix formation through antioxidant expression. This study reports plasma-enhanced chemical vapor deposition based amorphous silicon oxynitride (Si(ON)x) as a potential new fracture healing biomaterial that adheres well to the implant surface, releases Si(+4) to enhance osteogenesis, and forms a surface hydroxyapatite for collagen mineral attachment. These materials provide a sustained release of Si(+4) in physiological environment for extended times. The dissolution rate partially depends on the film chemistry and can be controlled by varying O/N ratio. The presence of Si(+4) enhances SOD1, which stimulates other osteogenic markers downstream and leads to rapid mineral formation. In vivo testing using a rat critical-sized calvarial defect model shows a more rapid bone-regeneration for these biomaterials as compared to control groups, that implies the clinical significance of the presented biomaterial.

Serotonin Inhibits Osteoblast Differentiation and Bone Regeneration in Rats.

Although increasing evidence indicates that serotonin (SER; 5-hydroxytrypamine [5-HT]) is involved in the regulation of bone metabolism, conflicting data exist regarding whether SER promotes or inhibits osteoblast differentiation and bone formation. Regeneration of functional bone is required for proper osseointegration of dental implants. Noticeably, the use of selective SER reuptake inhibitors was recently associated with the failure of osseointegrated dental implants. The present study examines the direct role of peripheral SER on the regulation of bone regeneration. The effect of SER on osteoblast differentiation and bone regeneration was examined using rat calvarial cell cultures in vitro and a rat critical-sized calvarial defect model in vivo. Rat calvarial cells expressed SER receptors Htr1 (5-HT1) and Htr2 (5-HT2), which are known to transmit signals in bone cells. In vitro, SER significantly reduced osteogenic differentiation and mineralization of rat calvarial cells with concomitant reduction of osteoblast marker genes including alkaline phosphatase (Alpl), osterix (Sp7), and osteocalcin (Bglap). Histologic and radiologic analyses using the rat critical-sized calvarial defect model revealed that the existence of SER significantly inhibited β-phase tricalcium phosphate-induced bone regeneration. Results suggest that SER in the local bone microenvironment might play a negative role in osteoblast differentiation and bone formation in rats.

Porous titanium scaffolds with injectable hyaluronic acid-DBM gel for bone substitution in a rat critical-sized calvarial defect model.

Demineralized bone matrix (DBM) is an allograft bone substitute used for bone repair surgery to overcome drawbacks of autologous bone grafting, such as limited supply and donor-site comorbidities. In view of different demineralization treatments to obtain DBM, we examined the biological performance of two differently demineralized types of DBM, i.e. by acidic treatment using hydrochloric acid (HCl) or treatment with the chelating agent ethylene diamine tetra-acetate (EDTA). First, we evaluated the osteo-inductive properties of both DBMs by implanting the materials subcutaneously in rats. Second, we evaluated the effects on bone formation by incorporating DBM in a hyaluronic acid (HA) gel to fill a porous titanium scaffold for use in a critical-sized calvarial defect model in 36 male Wistar rats. These porous titanium scaffolds were implanted empty or filled with HA gel containing either DBM HCl or DBM EDTA. Ectopically implanted DBM HCl and DBM EDTA did not induce ectopic bone formation over the course of 12weeks. For the calvarial defects, mean percentages of newly formed bone at 2weeks were significantly higher for Ti-Empty compared to Ti-HA+DBM HCl, but not compared to Ti-HA+DBM EDTA. Significant temporal bone formation was observed for Ti-Empty and Ti-HA+DBM HCl, but not for Ti-HA+DBM EDTA. At 8weeks there were no significant differences in values of bone formation between the three experimental constructs. In conclusion, these results showed that, under the current experimental conditions, neither DBM HCl nor DBM EDTA possess osteo-inductive properties. Additionally, in combination with an HA gel loaded in a porous titanium scaffold, DBM HCl and DBM EDTA showed similar amounts of new bone formation after 8weeks, which were lower than using the empty porous titanium scaffold. Copyright © 2016 John Wiley & Sons, Ltd.

Akermanite bioceramics promote osteogenesis, angiogenesis and suppress osteoclastogenesis for osteoporotic bone regeneration

It is a big challenge for bone healing under osteoporotic pathological condition with impaired angiogenesis, osteogenesis and remodeling. In the present study, the effect of Ca, Mg, Si containing akermanite bioceramics (Ca2MgSi2O7) extract on cell proliferation, osteogenic differentiation and angiogenic factor expression of BMSCs derived from ovariectomized rats (BMSCs-OVX) as well as the expression of osteoclastogenic factors was evaluated. The results showed that akermanite could enhance cell proliferation, ALP activity, expression of Runx2, BMP-2, BSP, OPN, OCN, OPG and angiogenic factors including VEGF and ANG-1. Meanwhile, akermanite could repress expression of osteoclastogenic factors including RANKL and TNF-α. Moreover, akermanite could activate ERK, P38, AKT and STAT3 signaling pathways, while crosstalk among these signaling pathways was evident. More importantly, the effect of akermanite extract on RANKL-induced osteoclastogenesis was evaluated by TRAP staining and real-time PCR assay. The results showed that akermanite could suppress osteoclast formation and expression of TRAP, cathepsin K and NFATc1. The in vivo experiments revealed that akermanite bioceramics dramatically stimulated osteogenesis and angiogenesis in an OVX rat critical-sized calvarial defect model. All these results suggest that akermanite bioceramics with the effects of Mg and Si ions on osteogenesis, angiogenesis and osteoclastogenesis are promising biomaterials for osteoporotic bone regeneration.

Mutation in Osteoactivin Decreases Bone Formation in Vivo and Osteoblast Differentiation in Vitro

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation invitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb(+)/SjJ (D2J/Gpnmb(+))]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb(+) mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. Invitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb(+) osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-β receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-β signaling. These data confirm the anabolic role of OA in postnatal bone formation.

Antibody-Mediated Osseous Regeneration: The Early Events in the Healing Response

Bone engineering strategies often exploit modulation of the extracellular environment, including delivery of cell and growth factors to repair and regenerate damaged tissues. During bone healing, the expression of endogenous bone morphogenetic proteins is an essential component of the healing response. However, in some situations, the inherent reparative capacity available in the local microenvironment is exceeded by the requirements of the defects. We have recently reported on a novel strategy, that exploits the specificity of antibodies to capture and make available endogenous osteogenic growth factors, referred to as "antibody-mediated osseous regeneration" (AMOR). The objective of the present study was to identify some of the cellular and molecular events involved in AMOR in an effort to begin to elucidate the mechanism of AMOR. The rat critical-sized calvarial defect model was used, where anti-bone morphogenetic protein (BMP)-2 monoclonal antibody (mAb), isotype-control mAb, or recombinant human (rh)BMP-2 were immobilized on absorbable collagen calvarial sponge (ACS) by adsorption, and then implanted into calvarial defects. The results demonstrated persistence of implanted mAbs for short term from 1 to 2 weeks after implantation. Increased cell infiltration was found in defects treated with anti-BMP-2 mAb. Examination of proteins on ACS scaffolds retrieved from defect sites demonstration increased levels of BMP-2, BMP-4, and BMP-7 proteins in sites implanted with anti-BMP-2 mAb. Moreover, BMP-2, BMP-4, and BMP-7 gene expression levels were increased in sites implanted with anti-BMP-2 mAb. Micro-computed tomography and histological analysis demonstrated that the bone within calvarial defects was fully regenerated in sites implanted with either anti-BMP-2 mAb or rhBMP-2. However, rhBMP-2-regenerated bone exhibited aberrant histomorphology with dystrophic calcification and invasion of subjacent areas. Altogether, the results revealed evidence for anti-BMP-2 mAbs to form an immune complex with BMP-2, BMP-4, and BMP-7, and bind to cells to mediate osteogenesis bone regeneration in vivo. This approach suggests a significant role for antibodies in regenerative orthopedic medicine.

In VivoOsteogenic Potential of Human Adipose-Derived Stem Cells/Poly Lactide-Co-Glycolic Acid Constructs for Bone Regeneration in a Rat Critical-Sized Calvarial Defect Model

<i>In Vivo</i>Osteogenic Potential of Human Adipose-Derived Stem Cells/Poly Lactide-Co-Glycolic Acid Constructs for Bone Regeneration in a Rat Critical-Sized Calvarial Defect Model

In VivoOsteogenic Potential of Human Adipose-Derived Stem Cells/Poly Lactide-Co-Glycolic Acid Constructs for Bone Regeneration in a Rat Critical-Sized Calvarial Defect Model

Recent studies suggest that human adipose tissue contains pluripotent stem cells, which are similar to bone marrow-derived stem cells. The objective of the present study was to assess the effect in bone regenerating capability of human adipose-derived stem cells (ADSCs) cultured in osteogenic media layered over poly lactide-co-glycolic acid (PLGA) and implanted in a critical nude rat calvarial defect. Twenty-seven nude rats were randomized into 3 groups (n = 9): 1) PLGA alone (control), 2) PLGA with undifferentiated ADSCs, and 3) PLGA with differentiated ADSCs. These 3 groups were divided into 9 subgroups (n = 3) according to in vitro pre-cultured periods (day 1 pre-culture (Group1), day 7 pre-culture (Group2), and day 14 pre-culture (Group3)) before implantation. An 8 mm critical-size circular calvarial defect was made in each nude rat. Specimens were harvested at 12 weeks post-implantation and evaluated radiographically and histologically. Radiodensitometric analysis revealed significantly higher bone growth in implants pre-cultured in osteogenic media for 14 days for Group 3. Histomorphometric analysis demonstrated that Groups 2 and 3 had bone formation filling 35% to 72% of the area of the defect after transplantation with cells that had been pre-cultured for 14 days. Constructs with differentiated ADSCs (Group 3) had noticeably more maximal and robust bone tissue regeneration than constructs with undifferentiated ADSCs (Group 2). These data provide evidence that constructs or implants made of PLGA and osteogenically differentiated ADSCs pre-cultured for 14 days before transplantation have better, more-robust bone regeneration capability in critical-sized skeletal defects than constructs with undifferentiated ADSCs. Human adipose derived stem cells can therefore be used as seed cells to construct tissue-engineered bone.