Critical-sized Rat Calvarial Defects Research Articles

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.

Porcine Bone Incorporated With 4-Hexylresorcinol Increases New Bone Formation by Suppression of the Nuclear Factor Kappa B Signaling Pathway.

The objectives of this study were to evaluate the suppression of the nuclear factor kappa B (NF-kB) pathway by 4-hexylresorcinol (4HR), which was activated by tumor necrosis factor-α (TNF-α) in osteoblasts, and new bone formation by 4HR-incorporated porcine bone in an animal model. For the confirmation of successful incorporation of 4HR into porcine bone, scanning electron microscopy (SEM) and Fourier transform-infrared (FT-IR) analysis were performed. High performance liquid chromatography was performed for the analysis of the 4HR release profile from porcine bone. MC 3T3-E1 cells were used for the analysis of the NF-kB signaling pathway activation by western blotting and real-time reverse transcriptase polymerase chain reaction. New bone formation and the analysis of marker protein expression were studied in a rat calvarial critical-sized defect model. Both SEM and FT-IR analysis demonstrated successful incorporation of 4HR into porcine bone. Approximately 30% of 4HR was steadily released from porcine bone for 18 days. 4HR suppressed the NF-kB signaling pathway, which was activated by TNF-α application in MC 3T3-E1 cells. Histological analysis revealed that porcine bone particles with incorporated 4HR showed significantly greater new bone formation than those without 4HR at 4 and 8 weeks after operation (P < 0.05). The expression intensities of alkaline phosphatase, osteoprotegerin, and osteocalcin were also higher in the 4HR-incorporated group. The application of 4HR suppressed the NF-kB signaling pathway in osteoblasts and 4HR-containing porcine bone particles promoted new bone formation in a rat calvarial defect model.

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.

Loading deproteinized bovine bone with strontium enhances bone regeneration in rat calvarial critical size defects

ObjectiveTo evaluate the effect of grafting with strontium (Sr)-loaded deproteinized bovine bone (DBB) on bone healing in calvarial critical size defects (CSD) in rats.Material and methodsTwo circular bone defects (5 mm in diameter) were created in the calvaria of 42 rats. One of the defects, randomly chosen, was grafted with (a) DBB, (b) DBB loaded with 19.6 μg/g of Sr (DBB/Sr1), or (c) DBB loaded with 98.1 μg/g of Sr (DBB/Sr2). The other defect was left empty as negative control. Groups of seven animals from each of the groups were euthanized 15 and 60 days post-op. Bone healing in the CSD was evaluated by micro-CT and histology/histomorphometry and immunohistochemistry.ResultsDBB/Sr2-grafted sites showed statistically significantly shorter radiographic residual defect length compared with DBB/Sr1- and DBB-grafted sites, and with empty controls at 60 days. Further, the amount of new bone formation in the DBB/Sr1- and DBB/Sr2-grafted sites was significantly higher compared with that in the DBB-grafted sites at 60 days. A larger number of DBB/Sr1- and DBB/Sr2-grafted sites presented with no- or only limited to mild inflammation, compared with the DBB-grafted sites, especially at 60 days. Higher expression of osteocalcin was observed in DBB/Sr1- and DBB/Sr2-grafted sites as compared to DBB-grafted sites.ConclusionGrafting with Sr-loaded DBB enhanced bone formation in CSD in rats, when compared with grafting with non-loaded DBB.Clinical relevanceGrafting with Sr-loaded DBB may enhance bone formation in bone defects.

Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.

The use of recombinant proteins has revolutionized the development of biologic pharmaceuticals; however, they are not free of complications. Some have very high molecular weight, some demonstrate in vivo instability, and the high cost of producing them remains a major problem. On the other hand, it has been shown that peptides derived from active domains keep their biologic activity and can trigger events, such as osteogenesis and bone regeneration. Small peptides are advantageous because of their ease of synthesis and handling and their low immunogenic activity. The purpose of this study was to investigate the functions of a synthetic peptide, cementum protein 1-peptide1 (CEMP-1-p1), both in vitro and in vivo. Our results show that CEMP-1-p1 significantly enhanced the proliferation and differentiation of human periodontal ligament cells toward a mineralizing-like phenotype, as evidenced by increasing alkaline phosphatase (ALP)-specific activity and osterix, runt-related transcription factor (RUNX)-2, integrin binding sialoprotein, bone morphogenetic protein-2, osteocalcin, and cementum protein (CEMP)-1 expression at mRNA and protein levels. In vivo assays performed through standardized critical-size calvarial defects in rats treated with CEMP-1-p1 resulted in newly formed bone after 30 and 60 d. These data demonstrate that CEMP-1-p1 is an effective bioactive peptide for bone tissue regeneration. The application of this bioactive peptide may lead to implementing new strategies for the regeneration of bone and other mineralized tissues.-Correa, R., Arenas, J., Montoya, G., Hoz, L., López, S., Salgado, F., Arroyo, R., Salmeron, N., Romo, E., Zeichner-David, M., Arzate, H. Synthetic cementum protein 1-derived peptide regulates mineralization in vitro and promotes bone regeneration in vivo.

3D-Plotted Beta-Tricalcium Phosphate Scaffolds with Smaller Pore Sizes Improve In Vivo Bone Regeneration and Biomechanical Properties in a Critical-Sized Calvarial Defect Rat Model.

Due to the difficulty in fabricating bioceramic scaffolds with smaller pore sizes by the current 3D printing technique, the effect of smaller pore sizes (below 400 µm) of 3D printed bioceramic scaffolds on the bone regeneration and biomechanical behavior is never studied. Herein beta-tricalcium phosphate (β-TCP) scaffolds with interconnected smaller pores of three different sizes (100, 250, and 400 µm) are fabricated by 3D plotting. The resultant scaffolds are then implanted into rat critical-sized calvarial defects without any seeded cells. A custom-designed device is developed to investigate the biomechanical properties of the scaffolds after surgical implantation for 4, 8, and 12 weeks. The scaffolds with the 100 µm pore size are found to present the highest maximum load and stiffness, comparable to those of the autogenous bone, after being implanted for 12 weeks. Micro-computed tomography (micro-CT) and histological analysis further indicate that the scaffolds with the 100 µm pore size achieve the highest percentage of new bone ingrowth, which correlates to their best in vivo biomechanical properties. This study demonstrates that tailoring the pore size of β-TCP scaffolds to a smaller range by 3D-plotting can be a facile and efficient approach to enhanced bone regeneration and biomechanical behaviors in bone repair.

Bone regeneration by freeze-dried composite of octacalcium phosphate collagen and teriparatide.

Octacalcium phosphate (OCP) and collagen (col) composite (OCPcol) demonstrated superior bone regeneration properties, and its commercialization appears to be forthcoming. As a practical medical material for new combination products, we developed a freeze-dried composite with OCPcol and teriparatide (TPTD) (OCPcolTPTDf), and investigated its bone regenerative properties. A disk of OCPcol was made by mixing OCP granules and atelocollagen for medical use. Then, OCPcolTPTDf was prepared by impregnation of the OCPcol disk with 1.0 or 0.1µg of TPTD solution (OCPcolTPTDf 1.0 and OCPcolTPTDf 0.1, respectively) followed by lyophilization. In vitro release profiles of TPTD from OCPcolTPTDf were determined using an enzyme-linked immunosorbent assay. Implantation of OCPcolTPTDf or OCPcol was carried out for a rat critical-sized calvarial defect. And five defects in each group were collected after 12weeks of implantation. The retention-release profiles of TPTD from OCPcolTPTDf supported a higher degree of retention of TPTD. Radiographic, histological, and histomorphometric examinations indicated that regenerated bone was filled in most of the defects of the OCPcolTPTDf. Additionally, the OCPcolTPTDf groups showed significantly enhanced bone regeneration compared with the OCPcol group. These results suggested that this newly developed bone regenerative composite could be a practical medical material.

Low power laser irradiation and human adipose-derived stem cell treatments promote bone regeneration of critical-sized calvarial defects in rats

The treatments of large bone defects are often accompanied by various limitations and complications. Stem cell therapy and physical treatments have both been shown to be beneficial in accelerating bone healing. However, the efficacy of combined treatment with stem cells and physical stimuli on large bone defects remains uncertain. The aim of this study was to evaluate the bone regeneration effects of low power laser irradiation (LPLI) and human ADSC treatments during fracture repair using a comparative rat calvarial defect model. The viability of human ADSCs was cultured on a porous PLGA scaffold using an MTS assay. The critical-sized (7 mm) calvarial bone defect Sprague–Dawley (SD) rats were divided into 4 groups: a control group, an LPLI group, an ADSC group, and an ADSC + LPLI group. Bone formation was evaluated using micro-CT at 4, 8, 12, and 16 weeks after surgery. New bone formation areas and osteogenic factor expression levels were then examined by histomorphological analysis and immunohistochemical staining. PLGA had no cytotoxic effect on human ADSCs. Micro-CT analyses showed that both the LPLI and ADSC groups improved calvarial bone defect healing compared to the control group. In addition, the ADSC + LPLI group showed significantly increased bone volume at 16 weeks after surgery. The area of new bone formation ranked as follows: control group < LPLI group < ADSC group < ADSC + LPLI group. There were significant differences between the groups. The ADSC and ADSC + LPLI groups showed strong signals of vWF expression in week 16. ADSC and LPLI treatments improve fracture repair in critical-sized calvarial defects in rats. Importantly, the combined treatment of ADSCs and further enhances the bone healing process.

RNA interfering molecule delivery from in situ forming biodegradable hydrogels for enhancement of bone formation in rat calvarial bone defects

RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12 weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. Statement of SignificanceDelivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-γ, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells.

Strontium-releasing fluorapatite glass-ceramic scaffolds: Structural characterization and in vivo performance

There is increasing interest in biodegradable ceramic scaffolds for bone tissue engineering capable of in situ delivery of ionic species favoring bone formation. Strontium has been shown to be osteogenic, but strontium-containing drugs such as strontium ranelate, used in Europe for the treatment of osteoporosis, are now restricted due to clinical evidence of systemic effects. By doping fluorapatite-based glasses with strontium, we developed ceramic scaffolds with fully interconnected macroporosity and cell size similar to that of cancellous bone, that are also capable of releasing strontium. The crystallization behavior, investigated by XRD and SEM, revealed the formation of akermanite and fluorapatite at the surface of strontium-free glass-ceramic scaffolds, and strontium-substituted fluorapatite at the surface of the strontium-doped scaffolds. At 8 weeks after implantation in a rat calvarial critical size defect, scaffolds doped with the highest amount of strontium led to the highest mineral apposition rate. A significantly higher amount of newly-formed bone was found with the strontium-free glass-ceramic scaffold, and possibly linked to the presence of akermanite at the scaffold surface. We demonstrate by energy dispersive XRF analyses of skull sections that strontium was present in newly formed bone with the strontium-doped scaffolds, while a significant amount of fluorine was incorporated in newly formed bone, regardless of composition or crystallization state. Statement of SignificanceThe present work demonstrates the in vivo action of strontium-containing glass-ceramic scaffolds. These bone graft substitutes are targeted at non load-bearing bone defects. Results show that strontium is successfully incorporated in newly formed bone. This is associated with a significantly higher Mineral Apposition Rate. The benefits of in situ release of strontium are demonstrated. The broader scientific impact of this works builds on the concept of resorbable ceramic scaffolds as reservoirs of ionic species capable of enhancing bone regeneration.

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.

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.

Epigallocatechin Gallate-Modified Gelatin Sponges Treated by Vacuum Heating as a Novel Scaffold for Bone Tissue Engineering.

Chemical modification of gelatin using epigallocatechin gallate (EGCG) promotes bone formation in vivo. However, further improvements are required to increase the mechanical strength and bone-forming ability of fabricated EGCG-modified gelatin sponges (EGCG-GS) for practical applications in regenerative therapy. In the present study, we investigated whether vacuum heating-induced dehydrothermal cross-linking of EGCG-GS enhances bone formation in critical-sized rat calvarial defects. The bone-forming ability of vacuum-heated EGCG-GS (vhEGCG-GS) and other sponges was evaluated by micro-computed tomography and histological staining. The degradation of sponges was assessed using protein assays, and cell morphology and proliferation were verified by scanning electron microscopy and immunostaining using osteoblastic UMR106 cells in vitro. Four weeks after the implantation of sponges, greater bone formation was detected for vhEGCG-GS than for EGCG-GS or vacuum-heated gelatin sponges (dehydrothermal cross-linked sponges without EGCG). In vitro experiments revealed that the relatively low degradability of vhEGCG-GS supports cell attachment, proliferation, and cell–cell communication on the matrix. These findings suggest that vacuum heating enhanced the bone forming ability of EGCG-GS, possibly via the dehydrothermal cross-linking of EGCG-GS, which provides a scaffold for cells, and by maintaining the pharmacological effect of EGCG.

Low power laser irradiation and human adipose-derived stem cell treatments promote bone regeneration in critical-sized calvarial defects in rats.

Both stem cell therapy and physical treatments have been shown to be beneficial in accelerating bone healing. However, the efficacy of combined treatment with stem cells and physical stimuli for large bone defects remains uncertain. The aim of this study was to evaluate the bone regeneration effects of low-power laser irradiation (LPLI) and human adipose-derived stem cell (ADSC) treatments during fracture repair using a comparative rat calvarial defect model. We evaluated the viability of human ADSCs, which were cultured on a porous PLGA scaffold using an MTS assay. The critical-sized calvarial bone defect rats were divided into 4 groups: control group, LPLI group, ADSC group, and ADSC+LPLI group. Bone formation was evaluated using micro-CT. New bone formation areas and osteogenic factor expression levels were then examined by histomorphological analysis and immunohistochemical staining. Our data showed that PLGA had no cytotoxic effect on human ADSCs. Micro-CT analyses revealed that both the LPLI and ADSC groups showed improved calvarial bone defect healing compared to the control group. In addition, the ADSC+LPLI group showed significantly increased bone volume at 16 weeks after surgery. The area of new bone formation ranked as follows: control group < LPLI group < ADSC group < ADSC+LPLI group. There were significant differences between the groups. In addition, both ADSC and ADSC+LPLI groups showed strong signals of vWF expression. ADSC and LPLI treatments improved fracture repair in critical-sized calvarial defects in rats. Importantly, the combined treatment of ADSCs and LPLI further enhances the bone healing process.

Effects of tissue processing on bioactivity of cartilage matrix-based hydrogels encapsulating osteoconductive particles

In the treatment of severe traumatic brain injury (TBI), decompressive craniectomy is commonly used to remove a large portion of calvarial bone to allow unimpeded brain swelling. Hydrogels have the potential to revolutionize TBI treatment by permitting a single-surgical intervention, remaining pliable during brain swelling, and tuned to regenerate bone after swelling has subsided. With this motivation, our goal is to present a pliable material capable of regenerating calvarial bone across a critical size defect. We therefore proposed the use of a methacrylated solubilized decellularized cartilage (MeSDCC) hydrogel encapsulating synthetic osteogenic particles of hydroxyapatite nanofibers, bioglass microparticles, or added rat bone marrow-derived mesenchymal stem cells (rMSCs) for bone regeneration in critical-size rat calvarial defects. Fibrin hydrogels were employed as a control material for the study. MeSDCC hydrogels exhibited sufficient rheological performance for material placement before crosslinking ( > 500 Pa), and sufficient compressive moduli post-crosslinking (E > 150 kPa). In vitro experiments suggested increased calcium deposition for cells seeded on the MeSDCC material; however, in vivo bone regeneration was minimal in both MeSDCC and fibrin groups, even with colloidal materials or added rMSCs. Minimal bone regeneration in the MeSDCC test groups may potentially be attributed to cartilage solubilization after decellularization, in which material signals may have degraded from enzymatic treatment. Looking to the future, an improvement in the bioactivity of the material will be crucial to the success of bone regeneration strategies for TBI treatment.

Superior calvarial bone regeneration using pentenoate-functionalized hyaluronic acid hydrogels with devitalized tendon particles

Traumatic brain injury (TBI) is a life-threatening condition defined by internal brain herniation. Severe TBI is commonly treated by a two-stage surgical intervention, where decompressive craniectomy is first conducted to remove a large portion of calvarial bone and allow unimpeded brain swelling. In the second surgery, spaced weeks to months after the first, cranioplasty is performed to restore the cranial bone. Hydrogels with paste-like precursor solutions for surgical placement may potentially revolutionize TBI treatment by permitting a single-stage surgical intervention, capable of being implanted with the initial surgery, remaining pliable during brain swelling, and tuned to regenerate calvarial bone after brain swelling has subsided. The current study evaluated the use of photocrosslinkable pentenoate-functionalized hyaluronic acid (PHA) and non-crosslinking hyaluronic acid (HA) hydrogels encapsulating naturally derived tissue particles of demineralized bone matrix (DBM), devitalized cartilage (DVC), devitalized meniscus (DVM), or devitalized tendon (DVT) for bone regeneration in critical-size rat calvarial defects. All hydrogel precursors exhibited a yield stress for placement and addition of particles increased the average material compressive modulus. The HA-DBM (4–30%), PHA (4%), and PHA-DVT (4–30%) groups had 5 (p < 0.0001), 3.1, and 3.2 (p < 0.05) times greater regenerated bone volume compared to the sham (untreated defect) group, respectively. In vitro cell studies suggested that the PHA-DVT (4–10%) group would have the most desirable performance. Overall, hydrogels containing DVT particles outperformed other materials in terms of bone regeneration in vivo and calcium deposition in vitro. Hydrogels containing DVT will be further evaluated in future rat TBI studies. Statement of SignificanceTraumatic brain injury (TBI) is a life-threatening condition characterized by severe brain swelling and is currently treated by a two-stage surgical procedure. Complications associated with the two-stage surgical intervention include the occurrence of the condition termed syndrome of the trephined; however, the condition is completely reversible once the secondary surgery is performed. A desirable TBI treatment would include a single surgical intervention to avoid syndrome of the trephined altogether. The first hurdle in reaching the overall goal is to develop a pliable hydrogel material that can regenerate the patient’s bone. The development of a pliable hydrogel technology would greatly impact the field of bone regeneration for TBI application and other areas of bone regeneration.

Biomimetic Composite Scaffold Containing Small Intestinal Submucosa and Mesoporous Bioactive Glass Exhibits High Osteogenic and Angiogenic Capacity.

Biomaterials with excellent osteogenic and angiogenic activities are desirable to repair massive bone defects. Decellularized matrix from porcine small intestinal submucosa (SIS) has attracted particular attention for tissue regeneration because it has strong angiogenic effects and retains plentiful bioactive components. However, it has inferior osteoinductivity and osteoconductivity. In this study, we developed porous composite of SIS combined with mesoporous bioactive glass (SIS/MBG) with the goal of improving the mechanical and biological properties. SIS/MBG scaffolds showed uniform interconnected macropores (∼150 μm), high porosity (∼76%), and enhanced compressive strength (∼0.87 MPa). The proliferation and osteogenic gene expression (Runx2, ALP, Ocn, and Col-Iα) of rat bone marrow stromal cells (rBMSCs) as well as the proliferation, angiogenic gene expression (VEGF, bFGF, and KDR), and tube formation capacity of human umbilical vein endothelial cells (HUVECs) in SIS/MBG scaffolds were significantly upregulated compared with nonmesoporous bioactive glass (BG)-modified SIS (SIS/BG) and SIS-only scaffolds. Western blot analysis revealed that SIS/MBG induced rBMSCs to osteogenic differentiation through the activation of Wnt/β-Catenin signaling pathway, and SIS/MBG enhanced angiogenic activity of HUVEC through the activation of PI3k/Akt pathways. The in vivo results demonstrated that SIS/MBG scaffolds significantly enhanced new bone formation and neovascularization simultaneously in critical-sized rat calvarial defects as compared with SIS/BG and SIS. Collectively, the osteostimulative and angiostimulative biomimetic composite scaffold SIS/MBG represents an exciting biomaterial option for bone regeneration.