Critical-sized Calvarial Defect Model Research Articles

Influences of donor and host age on human muscle-derived stem cell-mediated bone regeneration

BackgroundHuman muscle-derived stem cells (hMDSCs) have been shown to regenerate bone efficiently when they were transduced with Lenti-viral bone morphogenetic protein 2 (LBMP2). However, whether the age of hMDSCs and the animal host affect the bone regeneration capacity of hMDSCs and mechanism are unknown which prompted the current study.MethodsWe isolated three gender-matched young and old populations of skeletal muscle stem cells, and tested the influence of cells’ age on in vitro osteogenic differentiation using pellet culture before and after Lenti-BMP2/green fluorescent protein (GFP) transduction. We further investigated effects of the age of hMDSCs and animal host on hMDSC-mediated bone regeneration in a critical-size calvarial bone defect model in vivo. Micro-computer tomography (CT), histology, and immunohistochemistry were used to evaluate osteogenic differentiation and mineralization in vitro and bone regeneration in vivo. Western blot, quantitative polymerase chain reaction (PCR), and oxidative stress assay were performed to detect the effects of age of hMDSCs on cell survival and osteogenic-related genes. Serum insulin-like growth factor 1 (IGF1) and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured with an enzyme-linked immunosorbent assay (ELISA).ResultsWe found LBMP2/GFP transduction significantly enhanced osteogenic differentiation of hMDSCs in vitro, regardless of donor age. We also found old were as efficient as young LBMP2/GFP-transduced hMDSCs for regenerating functional bone in young and old mice. These findings correlated with lower phosphorylated p38MAPK expression and similar expression levels of cell survival genes and osteogenic-related genes in old hMDSCs relative to young hMDSCs. Old cells exhibited equivalent resistance to oxidative stress. However, both young and old donor cells regenerated less bone in old than young hosts. Impaired bone regeneration in older hosts was associated with high bone remodeling due to higher serum levels of RANKL and lower level of IGF-1.ConclusionhMDSC-mediated bone regeneration was not impaired by donor age when hMDSCs were transduced with LBMP2/GFP, but the age of the host adversely affected hMDSC-mediated bone regeneration. Regardless of donor and host age, hMDSCs formed functional bone, suggesting a promising cell resource for bone regeneration.

A polycaprolactone-β-tricalcium phosphate–heparan sulphate device for cranioplasty

BackgroundCranioplasty is a surgical procedure used to treat a bone defect or deformity in the skull. To date, there is little consensus on the standard-of-care for graft materials used in such a procedure. Graft materials must have sufficient mechanical strength to protect the underlying brain as well as the ability to integrate and support new bone growth. Also, the ideal graft material should be individually customized to the contours of the defect to ensure a suitable aesthetic outcome for the patient. PurposeCustomized 3D-printed scaffolds comprising of polycaprolactone-β-tricalcium phosphate (PCL-TCP) have been developed with mechanical properties suitable for cranioplasty. Osteostimulation of PCL-TCP was enhanced through the addition of a bone matrix-mimicking heparan sulphate glycosaminoglycan (HS3) with increased affinity for bone morphogenetic protein-2 (BMP-2). Efficacy of this PCL-TCP/HS3 combination device was assessed in a rat critical-sized calvarial defect model. MethodCritical-sized defects (5 mm) were created in both parietal bones of 19 Sprague Dawley rats (Male, 450–550 g). Each cranial defect was randomly assigned to 1 of 4 treatment groups: (1) A control group consisting of PCL-TCP/Fibrin alone (n = 5); (2) PCL-TCP/Fibrin-HSft (30 μg) (n = 6) (HSft is the flow-through during HS3 isolation that has reduced affinity for BMP-2); (3) PCL-TCP/Fibrin-HS3 (5 μg) (n = 6); (4) PCL-TCP/Fibrin-HS3 (30 μg) (n = 6). Scaffold integration and bone formation was evaluated 12-weeks post implantation by μCT and histology. ResultsTreatment with PCL-TCP/Fibrin alone (control) resulted in 23.7% ± 1.55% (BV/TV) of the calvarial defect being filled with new bone, a result similar to treatment with PCL-TCP/Fibrin scaffolds containing either HSft or HS3 (5 μg). At increased amounts of HS3 (30 μg), enhanced bone formation was evident (BV/TV = 38.6% ± 9.38%), a result 1.6-fold higher than control. Further assessment by 2D μCT and histology confirmed the presence of enhanced bone formation and scaffold integration with surrounding host bone only when scaffolds contained sufficient bone matrix-mimicking HS3. ConclusionEnhancing the biomimicry of devices using a heparan sulphate with increased affinity to BMP-2 can serve to improve the performance of PCL-TCP scaffolds and provides a suitable treatment for cranioplasty.

The Effects of Platelet-Derived Growth Factor-BB on Bone Marrow Stromal Cell-Mediated Vascularized Bone Regeneration.

Regenerative medicine for bone tissue mainly depends on efficient recruitment of endogenous or transplanted stem cells to guide bone regeneration. Platelet-derived growth factor (PDGF) is a functional factor that has been widely used in tissue regeneration and repair. However, the short half-life of PDGF limits its efficacy, and the mechanism by which PDGF regulates stem cell-based bone regeneration still needs to be elucidated. In this study, we established genetically modified PDGF-B-overexpressing bone marrow stromal cells (BMSCs) using a lentiviral vector and then explored the mechanism by which PDGF-BB regulates BMSC-based vascularized bone regeneration. Our results demonstrated that PDGF-BB increased osteogenic differentiation but inhibited adipogenic differentiation of BMSCs via the extracellular signal-related kinase 1/2 (ERK1/2) signaling pathway. In addition, secreted PDGF-BB significantly enhanced human umbilical vein endothelial cell (HUVEC) migration and angiogenesis via the phosphatidylinositol 3 kinase (PI3K)/AKT and ERK1/2 signaling pathways. We evaluated the effect of PDGF-B-modified BMSCs on bone regeneration using a critical-sized rat calvarial defect model. Radiography, micro-CT, and histological analyses revealed that PDGF-BB overexpression improved BMSC-mediated angiogenesis and osteogenesis during bone regeneration. These results suggest that PDGF-BB facilitates BMSC-based bone regeneration by enhancing the osteogenic and angiogenic abilities of BMSCs.

In situ silk fibroin-mediated crystal formation of octacalcium phosphate and its application in bone repair

The development of an ideal scaffold material is critical for the repair of bone defects. Being an important precursor of the mineralized matrix of bone tissue, octacalcium phosphate (OCP) has been considered a promising bone substitute. However, its application is largely limited due to the thermodynamical instability and poor processability of it. In this study, OCP was prepared by co-precipitation in the presence of small amount of silk fibroin (SF), which regulated the crystallization of OCP and led to the formation of SF-OCP complex. The diameters of OCP crystals in OCP, 0.1SF-OCP, 0.3SF-OCP and 1SF-OCP complexes were 489.0 ± 399.1 nm, 102.2 ± 50.7 nm, 94.7 ± 48.4 nm and 223.7 ± 167.6 nm, respectively. However, the shape of OCP crystals did not apparently change by the presence of SF. Further, porous SF/OCP composite scaffolds with pore size of 111.9 ± 33.1 μm were prepared, in which small crystals of SF-OCP complex were embedded in a SF matrix. MC3T3-E1 cells could attach and proliferate well on both the rugged surfaces and the pores of SF/OCP scaffolds, indicating their decent biocompatibility. Further, SF/OCP scaffolds markedly promoted bone regeneration in a rat calvarial critical-sized defect model. Both micro-CT and H&E characterizations showed that bone formation not only occurred around the scaffolds, but also penetrated into their center. Therefore, such SF/OCP composite scaffolds may have potential applications in bone tissue engineering.

Macroporous poly (l-lactic acid)/chitosan nanofibrous scaffolds through cloud point thermally induced phase separation for enhanced bone regeneration

A new method called cloud point thermally induced phase separation (CP-TIPS) was developed to prepare poly (l-lactic acid) (PLLA) scaffolds with macropores over 300 μm in PLLA/dioxane/H2O ternary system. The concentration of PLLA was 4% (w/v), and the solvent was dioxane with 14% of deionized water. By maintaining the clear solution of the ternary system near and above the cloud point for 2 h, followed by a large enough quenching depth (−80 °C), the crystallization of the α′-form crystal became privileged, and the microstructure of the pore wall was dominated with nanofibers. In contrast, conventional TIPS process resulted in scaffolds with similar pore size but platelet-like microstructure on the pore walls. The unique macroporous nanofibrous PLLA scaffold showed well elasticity and flexibility after swelling in 70% acetone. This property further enabled a facile “mechanical squeeze and intake” of high concentration viscous chitosan solution into the porous structure to modify the composite scaffolds. The compressive modulus of macroporous nanofibrous PLLA/CS scaffolds reached about 1 MPa. The hydrophilicity of the scaffold was greatly enhanced and the pH value remained stable in the first 3 weeks after degradation in vitro. The effect of chitosan concentration in the composite scaffolds on cell affinity and the osteogenic differentiation was evaluated. Finally, a critical-size rat calvarial bone defect model was used to evaluate the osteogenesis of the scaffolds in vivo and the composite scaffold with 3% chitosan significantly enhanced bone regeneration.

Preparation and osteogenic properties of poly ( L-lactic acid)/lecithin porous scaffolds with open pore structure

To investigate the preparation and osteogenic properties of poly ( L-lactic acid)(PLLA)/lecithin porous scaffolds with open pore structure. PLLA/lecithin porous scaffolds with different lecithin contents (0, 5%, 10%, 20%, 30%, 40%, 50%) were prepared by thermally induced phase separation (groups A, B, C, D, E, F, and G, respectively). Scanning electron microscopy (SEM) was used to observe the surface morphology of the scaffolds. Wide-angle X-ray diffraction (XRD) and differential scanning calorimetry (DSC) were used to detect the crystallinity of the scaffolds. The water uptake ability of the scaffolds was measured. The cell growth and viability of bone marrow mesenchymal stem cells (BMSCs) of mouse on each scaffold was assessed by cell counting kit 8 (CCK-8) method. The osteogenic differentiation ability of BMSCs on each scaffold was evaluated by alkaline phosphatase (ALP) activity. Finally, a critical-size rat calvarial bone defect model was used to evaluate the osteogenesis of the scaffolds in vivo. Micro-CT was used to reconstruct the three-dimensional model of the defect area, and the bone volume and bone mineral density were quantitatively analyzed. SEM results showed that the lecithin could slightly reduce the pore size; when lecithin content was 50%, platelet-like structure could be observed on the scaffolds. Wide angle XRD and DSC showed that the crystallinity of scaffolds gradually decreased with the increase of lecithin content. The water uptake ability test showed that the hydrophilicity of scaffolds increased with the increase of lecithin content. CCK-8 assay showed that cell activity gradually increased with the increase of culture time. After 7 days of culture, the absorbance ( A) value of groups C, D, E, and F were significantly higher than that of groups A, B, and G ( P<0.05), but no significant difference was found among groups C, D, E, and F ( P>0.05). After 14 days of osteogenic induction, with the increase of lecithin content, there was a significant difference in ALP activity of each group. The ALP activity in groups D, E, F, and G were significantly higher than that in groups A, B, and C ( P<0.05). In vivo, the results of Micro-CT examination and bone volume and bone mineral density showed that the scaffolds with 30% lecithin had the best repairing effect. Prepared by thermally induced phase separation, the cytocompatibility, osteogenic differentiation, and bone repair ability of the PLLA/lecithin porous scaffold is obviously better than that of pure PLLA scaffold. PLLA/lecithin porous scaffold with suitable lecithin content is a promising scaffold material for bone tissue engineering.

Phenotyping the Microvasculature in Critical-Sized Calvarial Defects via Multimodal Optical Imaging.

Tissue-engineered scaffolds are a powerful means of healing craniofacial bone defects arising from trauma or disease. Murine models of critical-sized bone defects are especially useful in understanding the role of microenvironmental factors such as vascularization on bone regeneration. Here, we demonstrate the capability of a novel multimodality imaging platform capable of acquiring in vivo images of microvascular architecture, microvascular blood flow, and tracer/cell tracking via intrinsic optical signaling (IOS), laser speckle contrast (LSC), and fluorescence (FL) imaging, respectively, in a critical-sized calvarial defect model. Defects that were 4 mm in diameter were made in the calvarial regions of mice followed by the implantation of osteoconductive scaffolds loaded with human adipose-derived stem cells embedded in fibrin gel. Using IOS imaging, we were able to visualize microvascular angiogenesis at the graft site and extracted morphological information such as vessel radius, length, and tortuosity two weeks after scaffold implantation. FL imaging allowed us to assess functional characteristics of the angiogenic vessel bed, such as time-to-peak of a fluorescent tracer, and also allowed us to track the distribution of fluorescently tagged human umbilical vein endothelial cells. Finally, we used LSC to characterize the in vivo hemodynamic response and maturity of the remodeled microvessels in the scaffold microenvironment. In this study, we provide a methodical framework for imaging tissue-engineered scaffolds, processing the images to extract key microenvironmental parameters, and visualizing these data in a manner that enables the characterization of the vascular phenotype and its effect on bone regeneration. Such multimodality imaging platforms can inform optimization and design of tissue-engineered scaffolds and elucidate the factors that promote enhanced vascularization and bone formation.

Hybrid scaffolds of Mg alloy mesh reinforced polymer/extracellular matrix composite for critical-sized calvarial defect reconstruction.

The challenge of developing scaffolds to reconstruct critical-sized calvarial defects without the addition of high levels of exogenous growth factor remains relevant. Both osteogenic regenerative efficacy and suitable mechanical properties for the temporary scaffold system are of importance. In this study, a Mg alloy mesh reinforced polymer/demineralized bone matrix (DBM) hybrid scaffold was designed where the hybrid scaffold was fabricated by a concurrent electrospinning/electrospraying of poly(lactic-co-glycolic acid) (PLGA) polymer and DBM suspended in hyaluronic acid (HA). The Mg alloy mesh significantly increased the flexural strength and modulus of PLGA/DBM hybrid scaffold. In vitro results demonstrated that the Mg alloy mesh reinforced PLGA/DBM hybrid scaffold (Mg-PLGA@HA&DBM) exhibited a stronger ability to promote the proliferation of bone marrow stem cells (BMSCs) and induce BMSC osteogenic differentiation compared with control scaffolding materials lacking critical components. In vivo osteogenesis studies were performed in a rat critical-sized calvarial defect model and incorporated a variety of histological stains and immunohistochemical staining of osteocalcin. At 12weeks, the rat model data showed that the degree of bone repair for the Mg-PLGA@HA&DBM scaffold was significantly greater than for those scaffolds lacking one or more of the principal components. Although complete defect filling was not achieved, the improved mechanical properties, promotion of BMSC proliferation and induction of BMSC osteogenic differentiation, and improved promotion of bone repair in the rat critical-sized calvarial defect model make Mg alloy mesh reinforced PLGA/DBM hybrid scaffold an attractive option for the repair of critical-sized bone defects where the addition of exogenous isolated growth factors is not employed.

Semaphorin 3A-stimulated bone marrow mesenchymal stem cells sheets promotes osteogenesis of type 2 diabetic rat

Objective: To evaluate the effect of semaphorin 3A (Sema3A) pre-treated bone marrow mesenchymal stem cells (BMSC) sheets on new bone formation in type 2 diabetes mellitus rats. Methods: Type 2 diabetes mellitus (T2DM) were induced by injection of streptozotocin, and the BMSC were isolated, controlled, identified and induced into cell sheets. Fifteen T2DM rats were randomly divided into control, sheets and Sema3A-sheets group and the calvarial critical size defect (CSD) model of rats were established. The defect zone of rats from control group were implanted with bone powder. The defect zone of rats from sheets group were implanted with bone powder and BMSC sheets. The defect zone of rats from Sema3A-sheets group were implanted with bone powder and BMSC sheets pretreated with 1.0 mg/L Sema3A. After 8 weeks, the bone samples were harvested and analyzed by micro-CT scanning, HE staining for the evaluation of new bone formation, and the immunohistochemical analysis for the expression of osteogenesis-related proteins including type Ⅰ collagen (COL- Ⅰ ), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN). Results: The BMSC were isolated and cultured, and oil red O and Alizarin red S staining proved the multi-potential differentiation. Eight weeks after the establishment of calvarial CSD model, Sema3A-sheet group showed the most abundant new bone formation (0.516±0.070), with increased bone volume fraction, namely bone volume/tissue volume (BV/TV) compared with sheets group (0.319±0.050) and control group (0.224±0.037) (P<0.05), and the sheets group showed increased BV/TV compared with control group (P<0.05). While trabecular thickness (Tb.Th) control group showed no difference in three groups (P>0.05). HE staining also confirmed that Sema3A-sheets group showed the most new bone formation. Sheet group (0.174±0.051) compared showed difference with control group (0.099±0.033) (P< 0.05), and Sema3A-sheet group (0.421±0.069) showed increased bone formation compared with sheet group and control group (P<0.05). Immunohistochemistry showed that BMSC sheet increased the expression of osteogenesis-related proteins including COL-Ⅰ, BMP-2 and OCN, while Sema3A pretreatment showed more obvious increase of the expression of COL-Ⅰ and OCN. Conclusions: The combined implantation of bone powder and Sema3A stimulated BMSC sheets significantly increased bone regeneration in vivo. Therefore, Sema3A pre-treated BMSC sheets transplantation provides a new strategy for restoring bone defect in T2DM.

Biomimetic mineralization of carboxymethyl chitosan nanofibers with improved osteogenic activity in vitro and in vivo

Inspired by the natural extracellular matrix, the organic-inorganic composite nanofibers are promising scaffolds for bone tissue engineering. Chitosan-based nanofibers are widely used as bone tissue engineering scaffolds with good biocompatibility but pungent solvents are frequently used for its processing. Carboxymethyl chitosan (CMCS), a water-soluble derivative of chitosan, has better biodegradability and bioactivity which allows CMCS to chelate Ca2+ and induce the deposition of apatite. Moreover, with water as solvent, CMCS nanofibers avoid the acidic salt removal comparing to electrospun-chitosan. In this study, we successfully prepared uniform CMCS nanofibers with the aid of polyethylene oxide (PEO) and obtained the optimized conditions with a voltage of 25 kV and PEO of molecular weight 1000 kDa. We further prepared hydroxyapatite (HA) coated electrospun CMCS nanofibers by biomimetic mineralization using 5 times simulated body fluid. The promotion of osteogenic differentiation of mouse bone marrow stromal cells (mBMSCs) in vitro was evaluated on the nanofibers scaffolds. Cell experiments revealed that CMCS-HA composite nanofibers increased the ALP activity. The gene expression level of Runx2 and ALP were about 1.6 and 4.3 folds at the 7 days, and 5.1 and 10 folds at the 14 days on CMCS-HA nanofibrous membranes than that on CMCS alone samples. The level of OCN increased by 24 and 1.5 times on the CMCS-HA scaffolds than CMCS scaffolds at the 14 and 21 days. In vivo new bone formation by nanofiber scaffolds was investigated in a critical-size rat calvarial bone defect model. Micro-CT results showed that the whole defect was covered by new bone after CMCS-HA filling the defect for 12 weeks. The results of H&E staining and Masson’s trichrome staining on histological sections further confirmed that composite nanofibers promoted new bone formation and maturation.

Guided osteoporotic bone regeneration with composite scaffolds of mineralized ECM/heparin membrane loaded with BMP2-related peptide.

IntroductionAt present, the treatment of osteoporotic defects poses a great challenge to clinicians, owing to the lower regeneration capacity of the osteoporotic bone as compared with the normal bone. The guided bone regeneration (GBR) technology provides a promising strategy to cure osteoporotic defects using bioactive membranes. The decellularized matrix from the small intestinal submucosa (SIS) has gained popularity for its natural microenvironment, which induces cell response.Materials and methodsIn this study, we developed heparinized mineralized SIS loaded with bone morphogenetic protein 2 (BMP2)-related peptide P28 (mSIS/P28) as a novel GBR membrane for guided osteoporotic bone regeneration. These mSIS/P28 membranes were obtained through the mineralization of SIS (mSIS), followed by P28 loading onto heparinized mSIS. The heparinized mSIS membrane was designed to improve the immobilization efficacy and facilitate controlled release of P28. P28 release from mSIS-heparin-P28 and its effects on the proliferation, viability, and osteogenic differentiation of bone marrow stromal stem cells from ovariectomized rats (rBMSCs-OVX) were investigated in vitro. Furthermore, a critical-sized OVX calvarial defect model was used to assess the bone regeneration capability of mSIS-heparin-P28 in vivo.ResultsIn vitro results showed that P28 release from mSIS-heparin-P28 occurred in a controlled manner, with a long-term release time of 40 days. Moreover, mSIS-heparin-P28 promoted cell proliferation and viability, alkaline phosphatase activity, and mRNA expression of osteogenesis-related genes in rBMSCs-OVX without the addition of extra osteogenic components. In vivo experiments revealed that mSIS-heparin-P28 dramatically stimulated osteoporotic bone regeneration.ConclusionThe heparinized mSIS loaded with P28 may serve as a potential GBR membrane for repairing osteoporotic defects.

Photobiomodulation of mesenchymal stem cells encapsulated in an injectable rhBMP4‐loaded hydrogel directs hard tissue bioengineering

Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering.

In Situ Bone Tissue Engineering With an Endogenous Stem Cell Mobilizer and Osteoinductive Nanofibrous Polymeric Scaffolds.

Classical bone tissue engineering involves the use of culture-expanded cells and scaffolds to produce tissue constructs for transplantation. Despite promising results, clinical adoption of these constructs has been limited due to various drawbacks, including extensive cell expansion steps, low cell survival rate upon transplantation, and the possibility of immuno-rejection. To bypass the ex vivo cell culture and transplantation process, the regenerative capacity of the host is exploited by mobilizing endogenous stem cells to the site of injury. Systemic injection of substance P (SP) induce mobilization of CD29+ CD105+ CD45- cells from bone marrow and enhance bone tissue regeneration in a critical-sized calvarial bone defect model. To provide an appropriate environment for endogenous stem cells to survive and differentiate into osteogenic lineage cells, electrospun nanofibrous polycaprolactone (PCL) scaffolds are functionalized with hydroxyapatite (HA) particles via a polydopamine (PDA) coating to create highly osteoinductive PCL-PDA-HA scaffolds that are implanted in defects. The combination of the PCL-PDA-HA scaffold and SP treatment enhance in situ bone tissue formation in defects. Thus, this in situ bone regeneration strategy, which combines recruitment of endogenous stem cells from the bone marrow to defective sites and implantation of a highly biocompatible and osteoinductive cell-free scaffold system, has potential as an effective therapeutic in regenerative medicine.

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.

Design and Characterization of a Therapeutic Non-phospholipid Liposomal Nanocarrier with Osteoinductive Characteristics To Promote Bone Formation.

Sterosomes are recently developed types of non-phospholipid liposomes formed from single-chain amphiphiles and high content of sterols. Although sterosomes presented significantly increased stability compared to conventional phospholipid liposomes, current sterosome biomaterials are not truly bioactive and have no intrinsic therapeutic effects. The purpose of this study was to develop a sterosome formulation with osteoinductive properties by an effective selection of sterol, one of the sterosome components. Oxysterols are oxidized derivatives of cholesterol and are known to stimulate osteogenesis and bone formation. Thus, 20S-hydroxycholesterol (Oxy), one of the most potent oxysterols for bone regeneration, was examined as a promising candidate molecule to form fluid lamellar phases with a single-chain amphiphile, namely, stearylamine (SA). First, the optimal composition was identified by investigating the phase behavior of SA/Oxy mixtures. Next, the capacity of the optimized SA/Oxy sterosomes to promote osteogenic differentiation of bone marrow stromal cells was assessed in vitro in a hydrogel environment. Furthermore, we explored the effects of osteogenic oxysterol sterosomes in vivo with the mouse critical-sized calvarial defect model. Our results showed that SA/Oxy sterosomes induced osteogenic differentiation in vitro and enhanced calvarial healing without delivery of additional therapeutic agents, indicating their intrinsic bone-forming potential. This study suggests a promising non-phospholipid liposomal platform with osteoinductive properties for delivery of small molecular drugs and/or other therapeutic genes for enhanced bone formation.

Structure-Performance Relationships of Temperature-Responsive PLGA-PEG-PLGA Gels for Sustained Release of Bone Morphogenetic Protein-2

PLGA (poly(lactic-co-glycolic) acid)-PEG (polyethylene glycol)-PLGA synthesis conditions have an impact on the physicochemical features of the copolymer and its usefulness as biomaterial. This study reports on an analysis of the composition and structural properties of PLGA-PEG-PLGA copolymers applying a variety of analytical techniques. Viscoelastic properties and particularly the temperature-responsive behavior of PLGA-PEG-PLGA showed a marked dependence on copolymer structural features. Physicochemical and biological properties, such as bioadhesion, biocompatibility and cell viability, of the raw copolymers and their gels were also evaluated. The most promising copolymer was chosen to formulate the osteoinductive protein bone morphogenetic protein-2 (125I-BMP-2), and the ability of its gels to sustain the release both in vitro and in vivo was monitored in situ using a gamma counter. In vitro diffusion studies were carried out using a bioinspired set-up that included a biorelevant receptor medium. In vivo release tests after implantation in a critical-size calvarial defect model showed an important burst, but then the release fitted well to the square-root kinetics. Importantly, the release rate constants recorded in vitro and in vivo matched each other suggesting close in vitro–in vivo correlation. Overall, the information gathered opens new perspectives in the biomedical application of these temperature-sensitive materials.

Extracellular calcium promotes bone formation from bone marrow mesenchymal stem cells by amplifying the effects of BMP-2 on SMAD signalling.

Understanding the molecular events that regulate osteoblast differentiation is essential for the development of effective approaches to bone regeneration. In this study, we analysed the osteoinductive properties of extracellular calcium in bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation. We cultured BM-MSCs in 3D gelatin scaffolds with Ca2+ and BMP-2 as osteoinductive agents. Early and late osteogenic gene expression and bone regeneration in a calvarial critical-size defect model demonstrate that extracellular Ca2+ enhances the effects of BMP-2 on Osteocalcin, Runx2 and Osterix expression and promotes bone regeneration in vivo. Moreover, we analysed the molecular mechanisms involved and observed an antagonistic effect between Ca2+ and BMP-2 on SMAD1/5, ERK and S6K signalling after 24 hours. More importantly, a cooperative effect between Ca2+ and BMP-2 on the phosphorylation of SMAD1/5, S6, GSK3 and total levels of β-CATENIN was observed at a later differentiation time (10 days). Furthermore, Ca2+ alone favoured the phosphorylation of SMAD1, which correlates with the induction of Bmp2 and Bmp4 gene expression. These data suggest that Ca2+ and BMP-2 cooperate and promote an autocrine/paracrine osteogenic feed-forward loop. On the whole, these results demonstrate the usefulness of calcium-based bone grafts or the addition of exogenous Ca2+ in bone tissue engineering.

Electrospun Silk Fibroin Nanofibrous Scaffolds with Two-Stage Hydroxyapatite Functionalization for Enhancing the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

The development of functional scaffolds with improved osteogenic potential is important for successful bone formation and mineralization in bone tissue engineering. In this study, we developed a functional electrospun silk fibroin (SF) nanofibrous scaffold functionalized with two-stage hydroxyapatite (HAp) particles, using mussel adhesive-inspired polydopamine (PDA) chemistry. HAp particles were first incorporated into SF scaffolds during the electrospinning process, and then immobilized onto the electrospun SF nanofibrous scaffolds containing HAp via PDA-mediated adhesive chemistry. We obtained two-stage HAp-functionalized SF nanofibrous scaffolds with improved mechanical properties and capable of providing a bone-specific physiological microenvironment. The developed scaffolds were tested for their ability to enhance the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and repair bone defect in vivo. To boost their ability for bone repair, we genetically modified hADMSCs with the transcriptional coactivator with PDZ-binding motif (TAZ) via polymer nanoparticle-mediated gene delivery. TAZ is a well-known transcriptional modulator that activates the osteogenic differentiation of mesenchymal stem cells (MSCs). Two-stage HAp-functionalized SF scaffolds significantly promoted the osteogenic differentiation of TAZ-transfected hADMSCs in vitro and enhanced mineralized bone formation in a critical-sized calvarial bone defect model. Our study shows the potential utility of SF scaffolds with nanofibrous structures and enriched inorganic components in bone tissue engineering.

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.

Hybrid composites of mesenchymal stem cell sheets, hydroxyapatite, and platelet-rich fibrin granules for bone regeneration in a rabbit calvarial critical-size defect model.

The reconstruction of large bone defects remains a major clinical challenge, and tissue engineering is a promising technique for resolving this problem. Many attempts have been made to optimize bone tissue engineering protocols. The aim of the present study was to develop a process incorporating mesenchymal stem cell (MSC) sheets with nanoscale hydroxyapatite (nano-HA) and autologous platelet-rich fibrin (PRF) granules for enhanced bone formation within a critical-sized rabbit cranial defect. MSC sheets and PRF were prepared prior to in vivo experiments. The osteogenic differentiation ability of MSCs and the ultrastructure of PRF were also studied. A total of 15 New Zealand white rabbits were used in the current study and critical-size defects (CSDs) were surgically introduced in the cranium (diameter, 15 mm). The surgical defects were treated with MSC/PRF composites, MSC composites or left empty. Animals were euthanized at week 8 post-surgery. Iconography, histological and histomorphometric analysis were performed to assess de novo bone formation. The percentage of new bone in the MSC/PRF group (35.7±5.1%) was significantly higher than that in the MSC (18.3±3.2%; P<0.05) and empty defect groups (4.7±1.5%; P<0.05). The results of the present study suggest that combined application of an MSC sheet with nano-HA and granular PRF enhances bone regeneration in a rabbit calvarial CSD model, and provides a novel insight into bone tissue regeneration for large bone defects.