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Early healing of bone defects is still a clinical challenge. Many bone-filling materials have been studied, among which photocrosslinked alginate has received significant attention due to its good biocompatibility and morphological plasticity. Although it has been confirmed that photocrosslinked alginate can be used as an extracellular matrix for 3D cell culture, it lacks osteogenesis-related biological functions. This study constructed a copper ions-photo dual-crosslinked alginate hydrogel scaffold by controlling the copper ion concentration. The scaffolds were shaped by photocrosslinking and then endowed with biological functions by copper ions crosslinking. According to in vitro research, the dual-crosslinked hydrogel increased the compressive strength and favored copper dose-dependent osteoblast differentiation and cell surface adherence of rat bone marrow mesenchymal stem cells and the expression of type I collagen (Col1), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), vascular endothelial growth factor (VEGF). In addition, hydrogel scaffolds were implanted into rat skull defects, and more angiogenesis and osteogenesis could be observed in in vivo studies. The above results show that the copper-photo-crosslinked hydrogel scaffold has excellent osseointegration properties and can potentially promote angiogenesis and early healing of bone defects, providing a reference solution for bone tissue engineering materials., (© 2024 Wiley Periodicals LLC.)

A combined biomaterial and cell-based solution to heal critical size bone defects in the craniomaxillofacial area is a promising alternative therapeutic option to improve upon autografting, the current gold standard. A shape memory polymer (SMP) scaffold, composed of biodegradable poly(ε-caprolactone) and coated with bioactive polydopamine, was evaluated with mesenchymal stromal cells (MSCs) derived from adipose (ADSC), bone marrow (BMSC), or umbilical cord (UCSC) tissue in their undifferentiated state or pre-differentiated toward osteoblasts for bone healing in a rat calvarial defect model. Pre-differentiating ADSCs and UCSCs resulted in higher new bone volume fraction (15.69% ± 1.64%) compared to empty (i.e., untreated) defects and scaffold-only (i.e., unseeded) groups (4.41% ± 1.11%). Notably, only differentiated UCSCs exhibited a significant increase in new bone volume, surpassing both undifferentiated UCSCs and unseeded scaffolds. Further, differentiated ADSCs and UCSCs had significantly higher trabecular numbers than their undifferentiated counterparts, unseeded scaffolds, and untreated defects. Although the mineral density regenerated within the unseeded scaffold surpassed that achieved with cell seeding, the connectivity of this bone was diminished, as the regenerated tissue confined itself to the spherical morphology of the scaffold pores. The SMP scaffold alone, with undifferentiated BMSCs, with undifferentiated and differentiated ADSCs, and differentiated UCSCs (29.72 ± 1.49 N) demonstrated significant osseointegration compared to empty defects (14.34 ± 2.21 N) after 12 weeks of healing when assessed by mechanical push-out testing. Based on these results and tissue availability to obtain the cells, pre-differentiated ADSCs and UCSCs emerge as particularly promising candidates when paired with the SMP scaffold for repairing critical size bone defects in the craniofacial skeleton., (© 2024 Wiley Periodicals LLC.)

Acid bone lysates (ABLs) represent the growth factors and other molecules released during autologous graft resorption. However, the impact of these bone-derived growth factors on the healing of bone defects has not yet been investigated. The aim of the present study was, therefore, to examine the impact of ABLs adsorbed to collagen membranes on bone regeneration. To this end, in 16 female Sprague Dawley rats, a standardized 5-mm-diameter critical size defect on the calvarial bone was created. The defects were covered with collagen membranes that had been soaked either in serum-free media or ABLs followed by lyophilization. After a healing period of 4 weeks, micro-computed tomography (μCT) and histological analyses by means of undecalcified thin ground sections were performed. μCT analysis of the inner 4 mm of the calvaria defect showed a greater bone defect coverage in the control group when compared to ABL group, 29.8% (confidence interval [CI]: 17.7-50.3) versus 5.6% (CI: 1.0-29.8, p = .03), respectively. Moreover, we found significantly more absolute bone volume (BV) in the control group when compared to ABL group, 0.59 mm 3 (CI: 0.27-1.25) versus 0.07 mm 3 (CI: 0.06-0.59, p = .04), respectively. Histomorphometry confirmed these findings with a relative BV in the central compartment of 14.1% (CI: 8.4-20.6) versus 5.6% (CI: 3.4-7.9, p = .004), respectively. These findings indicate that bone-derived growth factors contained in ABLs are able to attenuate bone regeneration within collagen membranes., (© 2020 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Cell sheet techniques are widely used in bone engineering. However, vascularization remains a challenge in fabricating vascularized engineered bone. The goal of this study was to induce adipose-derived stem cell (ADSC) osteogenic and angiogenic lineage differentiation and investigate the use of bidiretionally differentiated ADSCs for bone regeneration. ADSCs were cultured to form an osteogenic cell sheet. Other ADSCs were induced to differentiate into endothelial progenitor cells (EPCs), which were identified and characterized by morphological observation and CD31 immunofluorescent staining. Then, the ADSC sheet-EPC complexes were implanted subcutaneously into nude mice, while ADSC sheets alone were implanted as a control. After 8 weeks of transplantation, microcomputed tomography (micro-CT) and histological observation were used to assess bone formation. We then implanted the complexes in calvarial defects in rabbits and assessed bone repair by micro-CT and histological analysis. The ADSC sheets consisted of multiple layers of cells and extracellular matrix. The obtained EPCs formed capillary-like structures and expressed the specific antigen marker CD31. The osteogenic ADSC sheet-EPC complexes formed dense and well-vascularized new bone tissue at 8 weeks after implantation. Bone density was significantly lower in the control group than in the complex group (p < .05). In addition, the reconstruction of calvarial defects in rabbits in complex group was obviously greater than that in the control group (p < .05). These results suggested that the approach of engineering bone tissue with bidiretionally differentiated ADSCs enabled bone regeneration, thus offering a promising strategy for repairing bone defects., (© 2020 Wiley Periodicals, LLC.)

Adequate vascularization of scaffolds is a prerequisite for successful repair and regeneration of lost and damaged tissues. It has been suggested that the maturity of engineered vascular capillaries, which is largely determined by the presence of functional perivascular mural cells (or pericytes), plays a vital role in maintaining vessel integrity during tissue repair and regeneration. Here, we investigated the role of pericyte-supported-engineered capillaries in regenerating bone in a critical-size rat calvarial defect model. Prior to implantation, human umbilical vein endothelial cells and human bone marrow stromal cells (hBMSCs) were cocultured in a collagen hydrogel to induce endothelial cell morphogenesis into microcapillaries and hBMSC differentiation into pericytes. Upon implantation into the calvarial bone defects (8 mm), the prevascularized hydrogels showed better bone formation than either untreated controls or defects treated with autologous bone grafts (positive control). Bone formation parameters such as bone volume, coverage area, and vascularity were significantly better in the prevascularized hydrogel group than in the autologous bone group. Our results demonstrate that tissue constructs engineered with pericyte-supported vascular capillaries may approximate the regenerative capacity of autologous bone, despite the absence of osteoinductive or vasculogenic growth factors., (© 2021 John Wiley & Sons Ltd.)

Bone graft is required in various surgical procedures. Although autograft is the gold standard, it has limited availability. Various compounds have been proposed as alternatives such as biphasic calcium phosphate (BCP), which is the most widely used compound. The newly synthesized microporous sphere-shaped BCP has the advantage of increasing contact surface, and it can induce the formation of microbone structures. A putty-type contains the addition of a fluid carrier to the sphere-shaped BCP and can be easily used for a small orifice large bone defect. To compare the widely used BCP products, new bone formation and residual graft materials (RGM) were evaluated for 6 and 12 weeks in a rabbit calvarial bone defect model. Although existing BCP products and the microporous sphere-type product did not differ significantly with respect to new bone formation and RGM, the putty-type product was largely washed out and had low new bone formation at 6 and 12 weeks. Overall, the results suggest that microporous sphere-shaped BCP showed similar bone formation capability to existing products and was able to maintain higher initial mechanical stability., (© 2020 Wiley Periodicals LLC.)

Even though conventionally prepared octacalcium phosphate and collagen composite (OCP/Col) has exhibited excellent bone regeneration and has recently been commercialized for treating bone defects, reproducible appositional bone formation with OCP/Col has never been achieved. The present study investigated whether appositional bone formation could be achieved by altering the density of OCP/Col and applying liquid nitrogen during the preparation of OCP/Col. The prepared OCP/Col disks had eight variations and were divided into categories according to four different type of densities (1.0, 1.3, 1.7, and 2.0) of OCP/Col and two different pre-freezing conditions of gas phase (G group: -80°C) and liquid phase (L group: -196°C). These disks were implanted into subperiosteal pockets in rodent calvaria, five samples per each eight variations. Radiomorphometric analysis was conducted at 4 and 12 weeks after implantation, and histological analysis was conducted at 12 weeks after implantation. OCP/Col samples in the L group tended to retain their height and shape and had enhanced appositional bone formation, whereas OCP/Col samples in the G group tended to lose their height and shape and had limited appositional bone formation. The appositional bone formation increased along with growing density of OCP/Col, and L2.0 demonstrated higher appositional bone formation than other samples. These results suggest that the pre-freezing conditions and densities of OCP/Col affect the appositional bone formation., (© 2020 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc.)

In this study, the ability of silymarin to heal rat calvarial bone critical defects with mesenchymal stem cells isolated from human Wharton's jelly (HWJMSC) cultured on the electrospun scaffold of poly (lactic acid)/carbon nanotube (PLA/CNT) has been examined. In this study, 20 adult male Wistar rats were divided into four groups of five each. Under general anesthesia, 8 mm defects were created in the calvarial bone of the rats. Then, study groups were defined as no treatment group, the scaffold alone, the scaffold and HWJMSCs, and the scaffold/cells plus oral silymarin, respectively. The histomorphometric study was performed using H&E staining and Goldner's Masson trichrome as specific staining. The results of this study showed that the electrospun PLA/CNT scaffold is a biocompatible scaffold and HWJMSCs can considerably attach and proliferate on this scaffold, and the scaffold itself is also a suitable option for improving the bone repair process. The results of the histomorphometric analysis also showed a significantly higher amount of recently formed bone in the silymarin group plus scaffold/cells compared to the scaffold and cell group alone (p < .05). Utilizing silymarin plus HWJMSCs cultured on PLA/CNT scaffold can be used as a suitable method for the process of osteogenesis and bone repair., (© 2020 Wiley Periodicals, Inc.)

Material-induced ossification is suggested as a suitable approach to heal large bone defects. Fiber-reinforced composite-bioactive glasses (FRC-BGs) display properties that could enhance the ossification of calvarial defects. Here, we analyzed the healing processes of a FRC-BG implant in vivo from the perspective of material-induced ossification. Histological analysis of the implant, which was removed 5 months after insertion, showed the formation of viable, noninflammatory mesenchymal tissue with newly-formed mineralized woven bone, as well as nonmineralized connective tissue with capillaries and larger blood vessels. The presence of osteocytes was detected within the newly generated bone matrix. To expand our understanding on the osteogenic properties of FRC-BG, we cultured human adipose tissue-derived mesenchymal stromal cells (AD-MSCs) in the presence of two different BGs (45S5 and S53P4) and Al 2 O 3 control. AD-MSCs grew and proliferated on all the scaffolds tested, as well as secreted abundant extracellular matrix, when osteogenic differentiation was appropriately stimulated. 45S5 and S53P4 induced enhanced expression of COL2A1, COL10A1, COL5A1 collagen subunits, and pro-osteogenic genes BMP2 and BMP4. The concomitant downregulation of BMP3 was also detected. Our findings show that FRC-BG can support the vascularization of the implant and the formation of abundant connective tissue in vivo. Specifically, BG 45S5 and BG S53P4 are suited to evoke the osteogenic potential of host mesenchymal stromal cells. In conclusion, FRC-BG implant demonstrated material-induced ossification both in vitro and in vivo., (© 2020 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons Ltd.)

Our aims were to 1) evaluate the capacity of hollow hydroxyapatite (HA) microspheres (212-250 μm) to serve as a delivery system for controlled release of BMP-2 in vitro and 2) examine relaxin as an enhancer of BMP-2 for bone regeneration. Hollow HA microspheres were converted from borate glass microspheres and characterized using X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and the Brunauer-Emmett-Teller method. The microspheres loaded with BMP-2 and relaxin were implanted for 6 weeks in Sprague Dawley rats with calvarial defects. BMP-2 alone in the range up to 1 μg per defect exhibited dose-dependent bone regeneration while relaxin alone in the range up to 0.25 μg per defect did not promote bone regeneration. When compared with BMP-2 alone (1 μg per defect), a 50% reduction in the BMP-2 dose was achieved with the addition of 0.05, 0.1, or 0.25 μg of relaxin per defect. These results show that loading HA microspheres with a combination of relaxin and BMP-2 can significantly reduce the BMP-2 dose required to regenerate an equivalent amount of bone., (© 2020 Wiley Periodicals, Inc.)

The purpose of this study was to evaluate the glucose cross-linked gelatin/zein scaffolds for bone regeneration in vitro and in vivo. The nanofibrous scaffolds exhibited fast mineralization in the concentrated simulated body fluid with the deposited octacalcium phosphate and dicalcium phosphate dehydrate. The nanofibrous scaffolds exhibited no cytotoxic effect on MC3T3e1 cells in a CCK-8 test. Additionally, scanning electron microscope and confocal laser scanning microscopy images revealed that all the scaffolds were biocompatible and showed excellent support for MC3T3e1 cells. In the osteogenesis characterizations, Alizarin Red staining experiments indicated the improved calcium deposits on the cross-linked scaffolds, while the alkaline phosphatase activity showed no difference. Furthermore, the in vivo cranial bone regeneration results suggested that the cross-linked gelatin/zein scaffolds presented a strong positive effect on the cranial bone regeneration with the increased new bone volume and connective tissue formation, but the incorporation of zein in the gelatin scaffolds did not favor the bone regeneration. Moreover, the cross-linked gelatin scaffold retarded the bone resorption as indicated by the higher levels of IFN-γ and lower levels of IL-6, which restricted the differentiation of osteoclasts., (© 2019 Wiley Periodicals, Inc.)

Small molecules including sodium butyrate (SB) and dimethyloxalylglycine (DMOG) can promote bone regeneration via inhibitive effects eliciting cellular responses through signaling cascades. The purpose of this study was to determine the synergistic effects of SB and DMOG loaded on calcium sulfate (CaS) on bone regeneration in the challenging vertical augmentation model in the rabbit calvarium. Four plastic cylinders screwed on the calvarium of each of 10 rabbits were randomly grafted with CaS, CaS/SB, CaS/DMOG, or CaS/DMOG/SB. All specimens were assessed by radiographic, histologic, and histomorphometric analyses. In the radiographic analysis, three different layers (new bone, degraded CaS, and pristine CaS layers) could be distinguished within the cylinder in all groups at 2 weeks. Newly formed bone grew up from basal bone, and CaS in contact with newly formed bone was degraded into small particles to form a different layer. At 8 weeks, most of the pristine CaS had been absorbed and hardly seen within the cylinder. In the histomorphometric analysis, all groups showed comparable new bone areas and heights at 2 and 8 weeks. The DMOG group showed a significant increase in new bone area at 8 weeks compared with 2 weeks, but there was no significant difference among the groups at 8 weeks. The DMOG group showed significantly lower values for the residual material area than the control group at 2 weeks. Within the limitations of this study, SB and DMOG seem to exert smaller synergistic effects on bone regeneration compared to CaS alone in vertical bone augmentation., (© 2019 Wiley Periodicals, Inc.)

Hydroxyapatite/collagen (HAP/Col) composite has a nanostructure and composition similar to that of natural bone. Herein, we have evaluated the beneficial effects of acid-electrolyzed functional water (FW) in combination with HAP/Col composite as an irrigation material in a rat calvarium defect model. The rats were divided into four groups: control, PBS irrigation; FW, FW irrigation; HAP/Col, filled with HAP/Col; FW + HAP/Col, FW irrigation prior to HAP/Col filling. Bone volume (BV) and bone mineral density (BMD) of the newly formed bone were analyzed by microcomputed tomography. The results indicated that the combined use of FW and HAP/Col significantly augmented both BV (12.25 ± 1.93 mm 3 , control: 3.22 ± 0.55 mm 3 , 6 weeks) and BMD (120.09 ± 14.76 cm 3 /mg vs. control: 54.67 ± 7.20 cm 3 /mg, 6 weeks) in a time-dependent manner, which might be attributed to the soluble factor-inducing ability of FW. Based on this assumption, bFGF concentration in peripheral blood was measured. bFGF concentration was significantly increased in the FW + HAP/Col group (68.25 ± 9.2 pg/ml vs. control: 21.70 ± 8.18 pg/ml, 6 hr). Real-time PCR demonstrated significant augmentation of MCSF (2.82 ± 0.59-fold), RANKL (2.51 ± 0.33-fold) and BMP7 (1.66 ± 0.25-fold) (bone regeneration-related genes) and PDGF (1.31 ± 0.15-fold), VEGF (3.27 ± 0.42-ld) and IL-8 (6.77 ± 2.02-fold) (angiogenic genes) mRNAs in the FW + HAP/Col group. Taken together, these results suggest that the combined use of FW and HAP/Col induces bone regeneration, presumably by inducing the factors contributing to bone regeneration and angiogenesis., (© 2019 Wiley Periodicals, Inc.)

Bone defects are usually difficult to be regenerated due to pathological states or the size of the injury. Researchers are focusing on tissue engineering approaches in order to drive the regenerative events, using stem cells to regenerate bone. The purpose of this study is to evaluate the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) on biologically derived Gallus gallus domesticus-derived demineralized bone particle (GDD) sponge. The sponges were prepared by freeze-drying method using 1, 2, and 3 wt% GDD and cross-linked with glutaraldehyde. The GDD sponge was characterized using scanning electron microscopy, compressive strength, porosity, and Fourier transform infrared. The potential bioactivity of the sponge was evaluated by osteogenic differentiation of BMSCs using 3(4, dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and quantifying alkaline phosphatase (ALP) activity. in vivo experiments were evaluated through a micro-computerized tomography (μ-CT) and histological assays. The analysis confirmed that an increase in the concentration of the GDD in the sponge leads to a higher bone formation and deposition in rat calvarial defects. Histological assay results were in line with μ-CT. The results reported in this study demonstrated the potential application of GDD sponges as osteoinductor in bone tissue engineering in pathological or nonunion bone defects., (© 2020 John Wiley & Sons, Ltd.)

Here, we investigated the biocompatibility of a bioactive sodium calcium silicate glass containing 2.6 mol% Nb 2 O 5 (denoted BGPN2.6) and compare the results with the archetypal 45S5 bioglass. The glass bioactivity was tested using a range of in vitro and in vivo experiments to assess its suitability for bone regeneration applications. in vitro studies consisted of assessing the cytocompatibility of the BGPN2.6 glass with bone-marrow-derived mesenchymal stem cells (BM-MSCs). Systemic biocompatibility was verified by means of the quantification of biochemical markers and histopathology of liver, kidneys, and muscles. The glass genotoxicity was assessed using the micronucleus test. The regeneration of a calvarial defect was assessed using both qualitative and quantitative analysis of three-dimensional microcomputed tomography images. The BGPN2.6 glass was not cytotoxic to BM-MSCs. It is systemically biocompatible causing no signs of damage to high metabolic and excretory organs such as the liver and kidneys. No mutagenic potential was observed in the micronucleus test. MicroCT images showed that BGPN2.6 was able to nearly fully regenerate a critical-sized calvarial defect and was far superior to standard 45S5 Bioglass. Defects filled with BGPN2.6 glass showed over 90% coverage compare to just 66% for 45S5 Bioglass. For one animal the defect was completely filled in 8 weeks. These results clearly show that Nb-containing bioactive glasses are a safe and effective biomaterial for bone replacement., (© 2019 Wiley Periodicals, Inc.)

Carbonate apatite (CO 3 Ap) granules are known to show good osteoconductivity and replaced to new bone. On the other hand, it is well known that a porous structure allows bone tissue to penetrate its pores, and the optimal pore size for bone ingrowth is dependent on the composition and structure of the scaffold material. Therefore, the aim of this study was to fabricate various porous CO 3 Ap granules through a two-step dissolution-precipitation reaction using CaSO 4 as a precursor and 30-, 50-, 120-, and 205-μm diameter microfibers as porogen and to find the optimal pore size of CO 3 Ap. Porous CO 3 Ap granules were successfully fabricated with pore size 8.2-18.7% smaller than the size of the original fiber porogen. Two weeks after the reconstruction of rabbit calvarial bone defects using porous CO 3 Ap granules, the largest amount of mature bone was seen to be formed inside the pores of CO 3 Ap (120) [porous CO 3 Ap granules made using 120-μm microfiber] followed by CO 3 Ap (50) and CO 3 Ap (30). At 4 and 8 weeks, no statistically significant difference was observed based on the pore size, even though largest amount of mature bone was formed in case of CO 3 Ap (120). It is concluded, therefore, that the optimal pore size of the CO 3 Ap is that of CO 3 Ap (120), which is 85 μm., (© 2019 Wiley Periodicals, Inc.)

Modifications of sintering temperature and treatment time of bovine-derived bone grafts affect their physicochemical properties and further influence biological activity. Three different temperature sintered bovine-derived bone grafts: group I (300 °C 3 h), group II (300 °C 3 h plus 530 °C 6 h), and group III (300 °C 3 h plus 1000 °C 2 h) and Bio-Oss® were characterized and then compared in vitro for their effects on bone marrow stromal cells (BMSCs) migration, proliferation, and differentiation as estimated by cell migration assay, Alkaline phosphatase (ALP) activity assay, and Alizarin red staining. Further, the four bone grafts were implanted into the calvarial defects of rabbits to evaluate bone regeneration and graft degradation. The four deproteinized bovine-derived bone grafts displayed different surface topography. Group II displayed the highest potential of attracting cells. Both groups I and II markedly promote BMSCs differentiation. After 6 and 12 weeks, defects grafted with groups I and II displayed a significant higher bone fraction than defects grafted with group III and Bio-Oss®. Bone graft remnants remained in all four groups. Taken together, sintering at 300 °C for 3 h and sintering at 300 °C for 3 h with an addition of 530 °C for 6 h of bovine-dervied bone grafts displayed potential use in bone regeneration. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:272-281, 2020., (© 2019 Wiley Periodicals, Inc.)

Commercial bone substitute, such as calcined bovine bone (CBB), is currently extensively used as an alternative to autogenous bone. However, CBB lacks osteoinductivity and merely serves as a scaffold for native bone formation. To address this issue, we designed and prepared a heptaglutamate (E7)-modified BMP2-mimicking peptide (7E) and carried out a series of comprehensive physical characterizations and in vivo and in vitro studies to evaluate its role in the repair of cranial defects. The data elucidated that the amount of peptide anchoring to the bone graft materials was remarkably increased after modified with E7. Of note, 7E had a relatively stable and durable release, which promoted the osteogenic differentiation of rat derived bone marrow mesenchymal stem cells (BMSCs) and enhanced the bone regeneration of a rabbit calvarial defect by regulating the expression of the Runx2/SP7 axis. In summary, the composite biomaterials incorporating the E7-modified BMP2-mimicking peptide and CBB prepared in this study is a novel bone augmentation material with the merits of non-immunotoxicity, convenience, and low cost. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:80-93, 2020., (© 2019 Wiley Periodicals, Inc.)

Beta-tricalcium phosphate granular cement (β-TCP GC), consisting of β-TCP granules and an acidic calcium phosphate (Ca-P) solution, shows promise in the reconstruction of bone defects as it sets to form interconnected porous structures, that is, β-TCP granules are bridged with dicalcium phosphate dihydrate (DCPD) crystals. In this study, the effects of acidic Ca-P solution concentration (0-600 mmol/L) on the setting reaction and tissue response to β-TCP GC were investigated. The β-TCP GC set upon mixing with its liquid phase, based on the formation of DCPD crystals, which bridged β-TCP granules to one another. Diametral tensile strength of the set β-TCP GC was relatively the same, at ∼0.6 MPa, when the Ca-P concentration was 20-600 mmol/L. Due to the setting ability, reconstruction of the rat's calvarial bone defect using β-TCP GC with 20, 200, and 600 mmol/L Ca-P solution was much easier compared to that with β-TCP granules without setting ability. Four weeks after the reconstruction, the amount of new bone was the same, ∼17% in both β-TCP GC and β-TCP granules groups. Cellular response to β-TCP granules and β-TCP GC using the 20 mmol/L acidic Ca-P solution was almost the same. However, β-TCP GC using the 200 and 600 mmol/L acidic Ca-P solution showed a more severe inflammatory reaction. It is concluded, therefore, that β-TCP GC, using the 20 mmol/L acidic Ca-P solution, is recommended as this concentration allows surgical techniques to be performed easily and provides good mechanical strength, and the similar cellular response to β-TCP granules. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:22-29, 2020., (© 2019 Wiley Periodicals, Inc.)

The purpose of this study is to evaluate the efficacy of bone regeneration and volume maintenance of the three-dimensional (3D) structured biphasic calcium phosphate (BCP) block with porous hexahedron channels in a rabbit calvarial model. In this work, four circular defects (diameter: 8 mm) in calvarium of rabbits were randomly assigned to (1) negative control (control), (2) 3D hexahedron channel structured BCP block, (3) deproteinized bovine bone mineral particle, and (4) deproteinized porcine bone mineral particle. Animals were euthanized at 2 (n = 5) and 8 weeks (n = 5). Outcome measures included micro-computed tomography (CT) and histomorphometrical analysis. Results indicated that in micro-CT, BCP group showed the highest new bone volume with significant difference compared to control (p = 0.008) at 8 weeks. Histomorphometrically, total augmented area of BCP group was the highest with significant difference compared to control (p = 0.008) at 8 weeks. BCP group also maintained total volume of the original defect without collapsing. BCP block with 3D hexahedron channel structure seems to have favorable osteogenic and volume maintaining ability and highly porous structure might attribute to new bone formation. Further studies regarding the optimal internal structure and porosity of the BCP block bone substitute are needed. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2254-2262, 2019., (© 2019 Wiley Periodicals, Inc.)

Membranes are an integral component of guided bone regeneration protocols. This pre-clinical study was aimed at enhancing the bioactivity of collagen membranes by incorporating plasmid DNA (pDNA) or chemically modified RNA (cmRNA) encoding bone morphogenetic protein-9 (BMP-9). In addition, we also endeavored to harness the regenerative potential of the periosteum by creating perforations in the membrane. Nanoplexes of polyethylenimine (PEI)-nucleic acids (PEI-pDNA or PEI-cmRNA encoding BMP-9) were incorporated into commercially obtained and perforated collagen membranes (PCM) to produce PCM-pDNA(BMP-9) or PCM-cmRNA(BMP-9). After structural characterization, the biodegradation kinetics of PCM, PCM-pDNA(BMP-9) and PCM-cmRNA(BMP-9) were assessed in simulated body fluid in vitro. Using a 24-well transwell plate system with bone marrow stromal cells (BMSCs) in the lower chamber and the PCM to be tested in the upper chamber, the in vitro bioactivity of different PCMs was evaluated by measuring various markers for osteogenesis in BMSCs. Alkaline phosphatase activity was assessed in BMSCs, after 7 and 11 days of exposure to PCM, PCM-pDNA(BMP-9), or PCM-cmRNA(BMP-9). Similarly, calcium deposition and Alizarin red staining in BMSCs were assessed after 14 days of exposure to the three different types of PCM. PCMs were then tested in vivo using the calvarial defect model in rats. After 4 weeks, animals were euthanized and bone specimens were harvested for micro-computed tomography and histological assessments. Incorporation of pDNA or cmRNA did not alter the biodegradation profile of PCMs. Alkaline phosphatase activity trended toward being higher in BMSCs exposed to PCM-cmRNA(BMP-9) or PCM-pDNA(BMP-9), when compared to BMSCs alone. Similar trends were observed when calcium deposition and alizarin red staining was evaluated. Calvarial bone defects treated with PCM-cmRNA(BMP-9) resulted in significantly higher bone volume/total volume % (BV/TV%), when compared to empty defects and trended toward being higher than defects treated with PCM-pDNA(BMP-9) and PCM alone. We demonstrate for the first time that resorbable PCM can be utilized to efficiently deliver pDNA and cmRNA of interest. The released pDNA and cmRNA encoding BMP-9 in this assessment was shown to be functional in vitro as well as in vivo. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1824-1832, 2019., (© 2018 Wiley Periodicals, Inc.)

The hydrophilicity of bone graft material generally used as a carrier can play an important role in regulating bone morphogenetic protein (BMP) expression at the bone graft site. The hydrophilicity, altering physicochemical properties, and enhancing biological capabilities, can be increased via surface modification through ultraviolet (UV) photofunctionalization and the effect on de novo osteogenesis could be further improved. Therefore, this study aimed to assess the effects of UV-irradiated TiO 2 -coated hydroxyapatite (HA) in combination with rhBMP-2 on bone regeneration in rabbit calvarial defects. The hydrophilicity of HA and TiO 2 -coated HA pellets was evaluated by measuring the contact angle of water droplets with UV irradiation. To compare de novo osteogenesis in rabbit calvarial defects, the rabbits were segregated into four different groups: negative control, HA, TiO 2 -coated HA, and TiO 2 -coated HA with UV; histomorphometric analysis and micro-computed tomography (μCT) imaging were performed after 4 and 8 weeks. In vivo analysis revealed that de novo osteogenesis occurred on the critical size defects in all groups and was significantly increased in the TiO 2 -coated HA with UV group than in other groups (p < 0.05). The present results indicate that UV photofunctionalization promotes de novo osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1953-1959, 2019., (© 2018 Wiley Periodicals, Inc.)

The limitations of bone reconstruction techniques have stimulated the tissue engineering for the repair of large bone defects using osteoconductive materials and osteoinductive agents. This study evaluated the effects of low intensity electric current on the inorganic bovine graft in calvaria defects. Bone defects were performed with piezoelectric system in the calvaria of Wistar rats divided into four groups (n = 24): (C) without grafting and without electrical stimulation; (E) with grafting; (MC) without grafting and submitted to electrical stimulation; (MC + E) with grafting and submitted to electrical stimulation. Inflammatory, angiogenic and osteogenic events during bone repair at the 10th, 30th, 60th, and 90th days were considered. Several inflammatory markers demonstrated the efficacy of grafting in reducing inflammation, particularly when subjected to electrical stimulation. Angiogenesis and collagen organization were more evident by electrical stimulation application on the grafts. Moreover, the osteogenic cell differentiation process indicated that the application of microcurrent on grafting modulated the homeostasis of bone remodeling. It is concluded that microcurrent favored the performance of grafts in calvarial rat model. Low-intensity electrical current might improve the osteoconductive property of grafting in bone defects. Therefore, electrical current becomes an option as complementary therapy in clinical trials involving bone surgeries and injuries. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 924-932, 2019., (© 2018 Wiley Periodicals, Inc.)

Defect-specific bone regeneration using 3-dimensional (3D) printing of block bone has been developed. Polycaprolactone (PCL) is biocompatible polymer that can be used as 3D scaffold. The aim of this study is to assess the biocompatibility and osteogenic efficacy of 3D printed PCL scaffold and to evaluate the effectiveness of β-tricalcium phosphate (β-TCP) addition in PCL scaffold. In this work, four circular defects (diameter: 8 mm) in rabbit calvarium were randomly assigned to (1) negative control (control), (2) PCL block (PCL), (3) PCL mixed with 10 wt% β-TCP (PCL/β-TCP), and (4) PCL/β-TCP plus collagen membrane (PCL/β-TCP + M). Animals were euthanized at 2 (n = 5) and 8 weeks (n = 5). Results indicated that in micro-CT, PCL/β-TCP + M showed the highest total augmented volume and new bone volume at 8 weeks, but there was no significant difference among four groups. Histomorphometrically, PCL, PCL/β-TCP, and PCL/β-TCP + M showed the significantly higher total augmented area compared to the control. PCL/β-TCP + M showed the highest new bone area but not statistically higher than the control. New bone formation deep inside the scaffold was observed only in β-TCP added scaffold. PCL showed high biocompatibility with great volume maintenance. Addition of β-TCP to PCL seemed to increase hydrophilicity and osteoconductivity. Developments in 3D-printed PCL material are expected. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1254-1263, 2019., (© 2018 Wiley Periodicals, Inc.)

Restoring adequate blood supply is essential to the success of bone repair and augmentation procedures in craniofacial surgery. Nevertheless, the manner by which the incorporation of collagen gels (which can potentially induce angiogenesis), particulated deproteinized bovine bone grafts, or a combination of both can accelerate or delay bone regeneration in a clinical setting remains controversial. The objective of this study was to evaluate radiographically and histologically the capacity and functionality of particulated bone grafts and collagen gels on bone ossification and soft tissue formation in a rabbit calvarial defect. Bilateral calvarial defects in adult white New Zealand rabbits were filled or left either unfilled with bone grafts (DBBM), collagen gels (Gel), or a combination of both (DBBM + Gel). The defects were allowed to heal for 1, 2, and 6 months postoperatively before termination. Healing and regeneration patterns were assessed by 3D µCT and histological methods, and the biomechanical properties of regenerated tissue constructs were investigated and compared with autogenous calvarial bone. Results show that implanted DBBM and DBBM + Gel significantly enhanced immature bone formation compared with the empty and Gel groups; the latter treatment improved soft tissue formation and impeded immature bone formation but yielded no significant effect on mature bone formation. Implantation of DBBM not only effectively reconstructed 188.83 ± 25.25% of the tissue volume of the original defect, but it also regenerated bone tissue with similar tissue composition and biomechanical properties as the original autogenous bone. We also show that implanting different biomaterials can control the composition of soft and hard tissue in reconstructed tissue constructs in calvarial bone defects. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 529-544, 2019., (© 2018 Wiley Periodicals, Inc.)

The purpose of this study was to evaluate bone healing in calvarial defects using two bone graft substitute materials; biphasic beta-tricalcium phosphate/hydroxyapatite in hydrogel (ß-TCP/HA) versus composite non-demineralized xenogenic dentin with ß-TCP/HA mixture. Full thickness critical-sized defects were created bilaterally in 10 New Zealand male rabbits. Seven defects were left empty, six filled with biphasic tricalcium phosphate putty, and seven were filled with composite non-demineralized xenogenic dentin with biphasic tricalcium phosphate. Animals were sacrificed at eight weeks postoperatively and the healing of the biomaterial-filled defects was compared radiographically and by histomorphometry. Micro-computed tomography (μCT) was utilized to analyze the osteogenesis and healing patterns of the defects. Quantitative analysis of volume fraction (%) of the newly formed bone and remaining graft material (FV=filling volume/TV=tissue volume) and mean intensity [HU] in the defects were evaluated. Defects filled with composite dentin with biphasic tri-calcium phosphate showed volume fraction (FV/TV) in the order of 55.81% ± 17.72%, whereas defects filled with only biphasic tricalcium phosphate showed a fraction of 39.84% ± 16.06%, which represent the ratio of remaining graft material and new bone formation to the tissue volume. The empty negative control defects showed a volume fraction of 19.14% ± 8.787%. Histological analysis showed significant percentage increase in bone formation and residual graft with the composite Dentin/ß-TCP group after 8 weeks. The findings suggest that composite xenogenic dentin with biphasic tricalcium phosphate showed improved osteogenesis when compared to biphasic tricalcium phosphate without the addition of non-demineralized dentin. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 773-782, 2019., (© 2018 Wiley Periodicals, Inc.)

Effects of varied bioactive fillers on the biological behavior of porous polymer/inorganic composite scaffolds are lack of comprehensive comparison and remain elusive. Moreover, composite scaffolds with high porosity suffer from inferior mechanical performance. Herein, high-pressure molding and salt leaching were employed to prepare poly(ε-caprolactone) (PCL) composite porous scaffolds loaded with hydroxyapatite (HA) and bioactive glass (BG), respectively. Structural analysis indicated all the porous scaffolds presented interconnected open-pore structure with the porosity of ~87% and pore size of ~180 μm, hinging on the amounts and size of porogen. Compared to PCL/HA scaffolds, PCL/BG scaffolds showed ~2.3-fold augment in the water absorption. Attributing to the compact framework, the PCL/HA and PCL/BG porous scaffolds exhibited outstanding compressive modulus, which was notably higher than other PCL composite porous scaffolds reported in literatures. Cells culture results demonstrated that PCL/BG scaffolds displayed higher expression of osteogenic differentiation than PCL and PCL/HA scaffolds. Furthermore, in vivo results showed that more mature bone was formed within PCL/BG scaffolds than PCL/HA scaffolds, manifesting that the introduction of BG accelerated cranial bone regeneration to obtain complete bone healing within a short time. Therefore, these data indicate that PCL/BG scaffolds are more competitive for bone tissue engineering application. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 654-662, 2019., (© 2018 Wiley Periodicals, Inc.)

Bone morphogenetic protein-2 (BMP-2) is commonly used to enhance bone regeneration. The potential of BMP-2 for bone regeneration varies according to the concentration and release kinetics on the implanted site. Therefore, it is important to determine appropriate carriers of BMP-2. However, no optimal delivery vehicles have been identified. In the present study, we used alginate microbeads as a delivery vehicle for BMP-2. Alginate microbeads can be implanted onto the disease site through surgery or injection. The objective of this study was to evaluate that the osteoinductive properties of BMP-2 are effective in alginate microbeads as a carrier. In this study, the release kinetics of BMP-2 in alginate microbeads was evaluated using an enzyme-linked immunosorbent assay. BMP-2 released from alginate microbeads induced high alkaline phosphatase activity in canine adipose tissue-derived mesenchymal stem cells. Injection of alginate microbeads with BMP-2 into mouse subcutaneous tissue, as well as surgical implantation into the 5-mm circular calvarial defects in rats, was conducted and the results showed extensive new bone formation. In conclusion, alginate microbeads can be utilized as an effective BMP-2 delivery vehicle for use in orthopedic surgery and as an injectable vehicle for a minimally invasive therapy. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 286-294, 2019., (© 2018 Wiley Periodicals, Inc.)

Scaffolds implanted into bone defect sites must achieve optimal biodegradation rates while appropriately filling the void as new bone formation progresses. We recently developed a unique biomaterial consisting of salmon deoxyribose nucleic acid (DNA) and protamine, which can be used as an osteoconductive scaffold for tissue engineering. The aim of the present study was to elucidate how the degradation rate of the scaffold affects bone regeneration. We examined the relationships between the degradation rate of salmon DNA scaffolds and new bone formation using a rat skin flank subcutaneous model and rat calvarial defect model. The degradation rates of the scaffolds were proportional to the durations of pretreatment with ultraviolet (UV) light irradiation. The biodegradation rates of the scaffolds were also dependent on the duration of UV irradiation, as tested a subcutaneous tissue implantation. Scaffolds irradiated with UV light for 0.5 h maintained gradual biodegradation of phosphate compared with scaffolds irradiated for 0 or 3 h. In the calvarial defect model, we found that new bone formation was higher in rats treated with scaffolds irradiated with UV light for 0.5 h compared with those irradiated with UV light for 0 or 3.0 h. The present results suggest that bioengineering of scaffolds for biodegradation is important to regenerate bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 122-128, 2019., (© 2018 Wiley Periodicals, Inc.)

This study aimed to quantify the healing following vertical augmentation of allogenic bone blocks with/without recombinant human bone morphogenetic protein-2 (rhBMP-2) on rabbit calvaria. Experiments were performed using allogenic bone blocks which were grafted bilaterally with or without rhBMP-2 on 20 rabbit calvaria, and these animals were divided to four groups according to the use of rhBMP-2 and healing periods (2 and 8 weeks; n = 10 in each group). Onlay-type bone blocks (8 mm in diameter and 5 mm high) were fixed with a self-tapping screw after removing the cortex in the control group, and the same protocol was applied with the addition of soaking the bone blocks with rhBMP-2 for 15 min in the test group. Radiographic and histologic analyses were performed after 2 or 8 weeks to evaluate the volumetric stability and bone regeneration within the grafted area. The radiographic analysis revealed that the height of the allogenic bone block decreased but its volume was maintained from 2 to 8 weeks in both the control and test groups. The histologic results demonstrated a statistically significant increase in new bone area in the test group, especially in the lower region adjacent to the preexisting calvarial floor. The amount of newly formed bone in all regions of the augmented bone blocks in both the control and test groups was greater at 8 weeks than at 2 weeks. In conclusion, the vertically grafted allogenic bone block maintained its volume with new bone formation, and this was accelerated by the addition of rhBMP-2. These findings indicate that allogenic bone block soaked with rhBMP-2 could be a useful candidate biomaterial for vertical augmentation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2700-2707, 2018., (© 2018 Wiley Periodicals, Inc.)

Because guided bone regeneration (GBR) process is pronouncedly affected by the micro-environment in the defect, the surface stiffness of collagen membranes as a constituent part of the micro-environment was investigated in this study. The objective of this study was to manufacture biomimetically mineralized collagen membranes with controllable surface stiffness based on biomimetic strategy and to investigate the influences of surface stiffness on GBR process. The characterization and biocompatibility of membranes were examined in vitro. The mechanical properties of membranes were evaluated on macro and micro levels using tensile test and atomic force microscope, respectively. The critical-size cranial defect model and ectopic osteogenesis were chosen to employ their performances in vivo. The results indicated that the biomimetically mineralized collagen membranes with controllable surface stiffness were manufactured based on the biomimetic theory. The in vitro experiments showed that the mineralized collagen membrane with satisfactory surface stiffness can better promote the adhesion, proliferation, and osteogenic differentiation of mesenchymal stem cells. The membranes can perform excellently in both osteoinduction and osteoconduction, which results in effective manifestations in aspects of ectopic osteogenesis and GBR in vivo. Therefore, this biomimetically mineralized collagen membrane is a promising candidate for GBR treatment in future., (Copyright © 2018 John Wiley & Sons, Ltd.)

Octacalcium phosphate and collagen composite (OCP/Col) achieves stable bone regeneration without cell transplantation in preclinical studies. Recently, a sponsor-initiated clinical trial was conducted to commercialize the material. The present study investigated bone regeneration by OCP/Col with the single local administration of teriparatide (parathyroid hormone 1-34; TPTD). OCP/Col was prepared by mixing sieved granules of OCP and atelocollagen for medical use and a disk was molded. After the creation of a rodent critical-sized calvarial defect, OCP/Col or OCP/Col with dripped TPTD solution (1.0 or 0.1 µg; OCP/Col/TPTDd1.0 or OCP/Col/TPTDd0.1) was implanted into the defect. Six defects in each group were fixed 12 weeks after implantation. Radiographic examinations indicated that radiopaque figures in defects treated with OCP/Col with TPTD (OCP/Col/TPTDd) occupied a wider range than those treated with OCP/Col. Histological results demonstrated that most of the defect in OCP/Col/TPTDd was filled with newly formed bone. A histomorphometrical examination indicated that the percentage of newly formed bone was significantly higher in the defects of OCP/Col/TPTDd 1.0 (53.6 ± 4.3%) and OCP/Col/TPTDd 0.1 (52.2 ± 7.4%) than in those of OCP/Col (40.1 ± 8.4%), whereas no significant differences were observed between OCP/Col/TPTDd1.0 and OCP/Col/TPTDd0.1. These results suggest that OCP/Col with the single local administration of TPTD enhances bone regeneration in a rodent calvarial critical-sized bone defect. © 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1851-1857, 2018., (© 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc.)

In this study, an injectable and thermo-sensitive alginate/β-tricalcium phosphate hydrogel (TSAH/β-TCP) was prepared for aspirin release to a bone defect. Aspirin was dissolved into a mixture of poly(N-isopropylacrylamide) (PNIPAAm), an aminated alginate-g-PNIPAAm co-polymer, and β-TCP powders. Scanning electron microscopy showed that TSAH/β-TCP had an interconnected porous microstructure with a porosity of 86.78%. The cross-linked hydrogel released approximately 40% of the aspirin in the first 3 days and then slowly released the remainder. At a low concentration (≤100 μg/mL), aspirin did not promote cell proliferation, but enhanced the alkaline phosphatase activity, and osteocalcin (OCN) and collagen I expression of human bone marrow-derived mesenchymal stem cells. The TSAH/β-TCP/aspirin hydrogel was injected into the periosteum of the rat cranial bone, and its in vivo bone-forming ability was evaluated at 12 weeks. A bone morphogenetic protein 2 (BMP-2)-loaded TSAH/β-TCP hydrogel was injected as a control. Micro-computed tomography showed that the percentage of mineralized tissue in the TSAH/β-TCP/BMP-2 and TSAH/β-TCP/aspirin groups were similar and significantly higher than that in the TSAH/β-TCP group. Immunohistochemical staining showed considerable expression of OCN, especially in the TSAH/β-TCP/BMP-2 and TSAH/β-TCP/aspirin groups. These results suggest that the injectable TSAH/β-TCP/aspirin hydrogel has great potential for bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1739-1751, 2018., (© 2017 Wiley Periodicals, Inc.)

Recombinant human bone morphogenic protein (rhBMP) 9 has recently been reported to have more osteopromotive potential in vitro when compared to rhBMP2. The aim of the present study was to investigate the bone-inducing potential of rhBMP2 and rhBMP9. We compared rhBMP2, rhBMP7, and rhBMP9 at five different concentrations and showed convincingly that rhBMP9 possesses much greater potential for osteoblast differentiation even at 20 times lower concentrations in vitro. We further show that Noggin, an inhibitor for rhBMP2-induced osteogenesis, did not alter rhBMP9-induced osteogenesis. Thereafter, we show for the first time that rhBMP9 loaded onto atelo-collagen membranes is osteoinductive and has greater potential to form ectopic bone formation when compared to rhBMP2 even at four times lower doses. Similarly new bone formation of rhBMP2 and 9 when loaded on deproteinized bovine bone mineral (DBBM) was investigated in a rabbit calvarial defect. At 8 weeks, both rhBMP2 and rhBMP9 induced significantly higher new bone formation when compared to DBBM alone samples. Interestingly, once again four times lower dose of rhBMP9 group induced comparable or even greater levels of new bone height and new bone area when compared to the rhBMP2 group. The present study revealed that (1) rhBMP9 is capable of inducing ectopic new bone formation in vivo and (2) up to four times lower doses of rhBMP9 may be utilized to regenerate same-size bone defects when compared to rhBMP2. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1561-1574, 2018., (© 2018 Wiley Periodicals, Inc.)

Bone tissue engineering (BTE) holds promise for managing the clinical problem of large bone defects. However, clinical adoption of BTE is limited due to limited vascularization of constructs, which could be circumvented by pre-cultivation of osteogenic and endothelial derived cells in natural-based polymer scaffolds. However, until now not many studies compared the effect of mono- and cocultures pre-seeded in collagen before implantation. We utilized a mouse calvarial defect model and compared five groups of collagen scaffolds: a negative control of a collagen scaffold alone, a positive control treated with BMP-7, monocultures of either human osteoblasts (hOBs) or CD34+ cells, and a coculture of hOB and CD34 + cells. Each pre-seeded collagen scaffold was implanted in mice. After 6 weeks mice were sacrificed and their skulls prepared for volumetric and histologic analysis. We found that a monoculture of CD34 + cells and a coculture of hOB and CD34 + cells pre-cultured in the collagen scaffold increased bone regeneration to a similar extend. In these groups, greater amounts of new bone were found compared with hOB monocultures. Interestingly, monoculture of CD34 + cells demonstrated better fracture healing than monoculture of hOBs, emphasizing the possible role of angiogenesis. Our results are promising regarding a cellular based collagen BTE construct, but more work is needed to understand the complex interaction between the osteogenic and endothelial cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1505-1516, 2018., (© 2017 Wiley Periodicals, Inc.)

Extrusion free-forming, as a rapid prototyping technique, is extensively applied in fabricating ceramic material in bone tissue engineering. To improve the osteoinductivity of nano-hydroxyapatite (nHA) scaffold fabricated by extrusion free-forming, in this study, we incorporated a new peptide (P28) and optimized the superficial microstructure after shaping by controlling the sintering temperature. P28, a novel bone morphogenic protein 2 (BMP-2)-related peptide, was designed in this study. Analysis of the structure, physicochemical properties and release kinetics of P28 from nHA sintered at temperatures ranging from 1000 °C to 1400 °C revealed that nHA sintered at 1000 °C had higher porosity, preferable pore size and better capacity to control P28 release than that sintered at other temperatures. Moreover, the nHA scaffold sintered at 1000 °C with P28 showed improved adhesion, proliferation and osteogenic differentiation of MC3T3-E1 cells compared with scaffolds lacking P28 or BMP-2. In vivo, nHA scaffolds sintered at 1000 °C with P28 or BMP-2 induced greater bone regeneration in critical-sized rat cranial defects at 6 and 12 weeks post-implantation compared with scaffolds lacking P28 or BMP-2. Thus, nHA scaffolds sintered at 1000 °C and loaded with P28 may be excellent biomaterials for bone tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Bone defect reconstruction would be greatly improved if β-tricalcium phosphate (β-TCP) granules had the ability to self-set without sacrificing their osteoconductivity potential. This study aimed to identify a method to permit β-TCP self-setting whilst maintaining good osteoconductivity. When mixed with acidic calcium phosphate solution, β-TCP granules were found to readily set, forming a fully interconnected porous structure. On mixing, dicalcium phosphate dihydrate crystals formed on the surface of β-TCP granules, bridging the granules and resulting in the setting reaction. The setting time of the β-TCP granular cement (β-TCP GC) was approximately 1 min and its mechanical strength, in terms of diametral tensile strength, was approximately 0.8 MPa. The β-TCP GC and β-TCP granules both showed the same level of osteoconductivity within rat calvaria bone defects. At 2 and 4 weeks post-implantation, new bone formation was comparable between the two β-TCP based bone substitutes. We conclude that β-TCP GC has excellent potential for use as a cement in bone defect reconstruction. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 800-807, 2018., (© 2017 Wiley Periodicals, Inc.)

This study evaluated the sustained release effect of clarithromycin-loaded in PLGA microspheres in a rabbit calvaria defect model. Four bone defects (ø5.0) were created in the calvaria of New Zealand White rabbits (n = 21, n = 7/time point). The defects were randomly designated to four groups. Group 1: No augmentation (sham), Group 2: beta-tricalcium phosphate (β-TCP), Group 3: β-TCP with 0.12 µg clarithromycin, and Group 4: β-TCP with 6.12 µg PLGA microspheres loaded with 0.12 µg Clarithromycin. After 2, 4, and 12 weeks of healing, bone regeneration was evaluated using micro-computed tomography (µCT) and histology. Clarithromycin release from PLGA microspheres revealed sustained release for around 4 weeks with ∼50% release during the first week. Histologically, new bone formation was evident at 2 and 4 weeks of healing in all groups and bone formation increased as a function of healing time. At 12 weeks, Group 4 showed significantly higher amount of newly formed bone compared to Group 1. The µCT showed that Group 4 expressed significantly higher bone formation compared to Group 1 at all time points. The in vivo findings showed that β-TCP with clarithromycin-loaded microspheres can enhance bone formation in bone defects. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 201-208, 2018., (© 2017 Wiley Periodicals, Inc.)

Salt-leaching using powder (SLUP) scaffolds are novel salt-leaching scaffolds with well-interconnected pores that do not require an organic solvent or high pressure. In this study, in vitro and in vivo cell behaviors were assessed using a PCL (polycaprolactone) SLUP scaffold. Moreover, using PCL, conventional salt-leaching and 3D-plotted scaffolds were fabricated as control scaffolds. Morphology, mechanical property, water absorption, and in vitro/in vivo cell response assessments were performed to clarify the characteristics of the SLUP scaffold compared with control scaffolds. Consequently, we verified that the interconnectivity between the pores of the SLUP scaffold was enhanced compared with conventional salt-leaching scaffolds. Moreover, in vitro cell attachment and proliferation of the SLUP scaffold were higher than those of the 3D-plotted scaffold because of their morphological characteristic. Furthermore, we revealed that new bone formation and bone ingrowth of the SLUP scaffold was superior to those of the calvarial defect model and 3D-plotted scaffold because of the high porosity and improved interconnectivity of pores by the SLUP technique without high pressure using powders. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3432-3444, 2017., (© 2017 Wiley Periodicals, Inc.)

Mastoidectomy, the removal of infected mastoid bones, is a common surgical procedure for the treatment of chronic otitis media. Persistent and recurrent otorrhea and accumulation of keratin debris following open cavity mastoidectomy are still bothersome issues for both patients and otologists. In this study, we used human ear adipose-derived stromal cells (hEASCs) in combination with polycaprolactone (PCL) scaffolds and osteogenic differentiation medium (ODM) to regenerate temporal bone defects. The hEASCs showed stem cell phenotypes, and these characteristics were maintained up to passage 5. Mastoid bulla and cranial bone defects were induced in Sprague-Dawley rats using AgNO 3 and burr hole drilling, respectively, and the rats were then divided into five groups: (1) control, (2) hEASCs, (3) hEASCs + ODM, (4) hEASCs + PCL scaffolds, and (5) hEASCs + PCL scaffolds + ODM. Osteogenesis was evaluated by micro-computed tomography and histology. Compared with the control group, the groups transplanted with hEASCs and PCL scaffolds had significantly higher bone formation along the periphery of the mastoid bulla area. Moreover, ODM synergistically enhanced bone formation in mastoid bulla defects. Our results suggest that combining hEASCs with PCL scaffolds represents a promising method for anatomical and functional reconstruction of postoperative temporal bone defects following mastoidectomy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3493-3501, 2017., (© 2017 Wiley Periodicals, Inc.)

Repairing large calvarial bone defects remains a challenging task. Previously, it was discovered that that miR-148b, when acting in concert with bone morphogenetic protein 2 (BMP-2), enhanced the osteogenesis of human adipose-derived stem cells (hASCs) and improved calvarial bone healing in nude mice. However, the molecular target of miR-148b remained elusive. Here it is revealed that miR-148b directly targets NOG, whose gene product (noggin) is an antagonist to BMPs and negatively regulates BMP-induced osteogenic differentiation and bone formation. A new Cre/loxP-based baculovirus system was employed to drive prolonged BMP-2 and miR-148b overexpression in hASCs, wherein the BMP-2 overexpression induced noggin expression but the concurrent miR-148b expression downregulated noggin, thus relieving the negative regulatory loop and ameliorating hASC osteogenesis without hindering hASC proliferation or triggering appreciable cytotoxicity. Implantation of the engineered hASCs coexpressing BMP-2 and miR-148b into nude mice enabled substantial repair of critical-size calvarial bone defects (4 mm diameter) at 12 weeks post-transplantation, filling 83% of the defect area, 75% of bone volume and restoring the bone density to 89% of the original bone density. Such superior healing effects indicate the potential of the Cre/loxP-based baculovirus-mediated BMP-2/miR-148b expression for calvarial bone repair. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

This study assesses the biocompatibility of novel silk protein membranes with and without modification, and evaluates their effect on facilitating bone formation and defect repair in guided bone regeneration. Two calvarian bone defects 12 mm in diameter were created in each of a total of 38 rabbits. Four different types of membranes, (silk-, hydroxyapatite-modified silk-, β-TCP-modified silk- and commonly clinically used collagen-membranes) were implanted to cover one of the two defects in each animal. Histologic analysis did not show any adverse tissue reactions in any of the defect sites indicating good biocompatibility of all silk protein membranes. Histomorphometric and histologic evaluation revealed that collagen and β-TCP modified silk membranes supported bone formation (collagen: bone area fraction p = 0.025; significant; β-TCP modified silk membranes bone area fraction: p = 0.24, not significant), guided bone regeneration and defect bridging. The bone, which had formed in defects covered by β-TCP modified silk membranes, displayed a more advanced stage of bone tissue maturation with restoration of the original calvarial bone microarchitecture when compared to the bone which had formed in defects, for which any of the other test membranes were used. Micro-CT analysis did not reveal any differences in the amount of bone formation between defects with and without membranes. In contrast to the collagen membranes, β-TCP modified silk membranes were visible in all cases and may therefore be advantageous for further supporting bone formation beyond 10 weeks and preventing soft tissue ingrowth from the periphery. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2603-2611, 2017., (© 2016 Wiley Periodicals, Inc.)

Recent research has demonstrated that recombinant human bone morphogenetic protein 9 (rhBMP9) has been considered the most osteoinductive growth factor of the BMP-family. In the present study, rhBMP9 was investigated for its influence in combination with two biomaterials for bone regenerative medicine. Either porcine-derived collagen membrane (CM) or deproteinized bovine bone mineral (DBM) combined with 20 µg of rhBMP9 were implanted in 6 mm rabbit calvarial defects. Bone augmentation was evaluated by microCT and histomorphometry at 8 weeks post-surgery. Both CM + rhBMP9 and DBM + rhBMP9 groups significantly promoted mineralized tissue volume (microCT) and area, new bone height and area (histomorphometric measurements) when compared to CM and DBM alone groups or control (empty). All specimens in the CM + rhBMP9 group but not all in the DBM + rhBMP9 group induced a complete horizontal bone defect closure. Multinucleated giant cells (MNGCs) were observed directly in contact with DBM surfaces irrespective of rhBMP9, whereas CM was generally not associated to the presence of MNGCs. When combined with rhBMP9, DBM augmented a larger volume of mineralized tissue (including the mineralized bone graft), whereas CM induced greater volume of native host bone. While DBM in combination with rhBMP9 induced higher mineralized tissue mostly associated with the bone grafting material, CM may have presented preferable results based on a higher horizontal defect closure with a faster regeneration of host new bone. The effect of including collagen within the carrier system of rhBMP9 on bone regeneration justifies further evaluation of this combination procedure in larger animal models. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2655-2661, 2017., (© 2017 Wiley Periodicals, Inc.)

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 12 weeks. For the calvarial defects, mean percentages of newly formed bone at 2 weeks 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 8 weeks 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 8 weeks, which were lower than using the empty porous titanium scaffold. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The present study was designed to investigate whether composite of coprecipitating octacalcium phosphate and gelatin (C-OCP/Gel) has an effect in repairing critical-sized defect of rat calvaria with oriented regenerative bone if implanted. The materials were prepared through two steps to disperse homogenous and well-elongated OCP toward long axis of the crystals in gelatin (Gel) matrix with the distinct concentration 17-44 wt %: OCP precipitates recovered from the coprecipitated with Gel molecules in aqueous solution (referred to as C-OCP hereafter) were mixed again in fresh aqueous Gel solution with various mixing ratio to form C-OCP/Gel for implantation. C-OCP/Gel disks with 9 mm diameter and 1 mm thickness after the dehydrothermal treatment was implanted in 9 mm diameter rat calvaria critical-sized defect. The histology, the histomorphometry in the regenerated bone and the quantitative analysis of the orientation of collagen with picrosirius red staining were carried out. It was found that C-OCP/Gel is capable of not only inducing sufficiently regenerative bone over 80% of the defect coupled with practically complete material biodegradation but also forming oriented bone significantly in relation to the amount of C-OCP in Gel matrix until 12 weeks after the implantation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1029-1039, 2017., (© 2016 Wiley Periodicals, Inc.)

Bioactive glass has been demonstrated as a biocompatible bone substitute. However bone healing process can be prolonged due to late resorption of the material. Adipose derived stem cells (ASC) have osteogenic differentiation potential and hence can be a cell source for bone regeneration. The aim of this study was to test whether combination of bioactive glass with ASCs would enhance bone regeneration. Following creation of critical sized defects on the calvaria of 32 Wistar rats, the animals were randomly divided into four groups: Group C (control): Defects were left untreated; Group G: Defects were covered with autologous bone graft; Group BG: Defects were filled with bioactive glass; Group BG/ASC: Defects were filled with bioactive glass seeded with ASCs. The defect size was significantly greater in Group C compared to all other groups. Bone density was significantly lower in Group C compared to Group G and Group BG/ASC. Bone regeneration score of Group C was significantly lower than other groups. Group BG/ASC demonstrated lamellar bone and havers canal formation. The results of this study demonstrated that bioactive glass implanted with ASC is a biocompatible construct stimulating radiologically and histologically evident bone regeneration similar to autologous bone grafting. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1002-1008, 2017., (© 2016 Wiley Periodicals, Inc.)

Bone has the potential for spontaneous healing. This process, however, often fails in patients with comorbidities. Tissue engineering combining functional cells, biomaterials and osteoinductive cues may provide alternative treatment strategies. We have recently demonstrated that stromal cell-derived factor-1β (SDF-1β) works in concert with bone morphogenetic protein-2 (BMP-2) to potentiate osteogenic differentiation of bone marrow-derived mesenchymal stem/stromal cells (BMSCs). Here, we test the hypothesis that SDF-1β overexpressed in Tet-Off-SDF-1β BMSCs, delivered on acellular dermal matrix (ADM), synergistically augments BMP-2-induced healing of critical-sized mouse calvarial defects. BMSC therapies alone showed limited bone healing, which was increased with co-delivery of BMP-2. This was further enhanced in Tet-Off-SDF-1β BMSCs + BMP-2. Only limited BMSC retention on ADM constructs was observed after 4 weeks in vivo, which was increased with BMP-2 co-delivery. In vitro cell proliferation studies showed that supplementing BMP-2 to Tet-Off BMSCs significantly increased the cell number during the first 24 h. Consequently, the increased cell numbers decreased the detectable BMP-2 levels in the medium, but increased cell-associated BMP-2. The data suggest that SDF-1β provides synergistic effects supporting BMP-2-induced, BMSC-mediated bone formation and appears suitable for optimization of bone augmentation in combination therapy protocols. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Both of the osteogenic factor and the suitable delivery system in bone tissue engineering are essential for bone regeneration. In this study, we manufactured two kinds of composite vehicles for sustained release of rhBMP-2 and its derived synthetic oligopeptide (Peptide-24, abbreviated as P24) for osteogenesis and bone defect repair. The composite vehicle was based on cross-linked chitosan, nano-hydroxyapatite/collagen (nHAC), and poly (lactide-co-glycolide) acid microsphere. The physicochemical properties of the composite vehicles (abbreviated as CS/nHAC/PLGA-MS) were investigated. The rhBMP-2 and P24 release kinetics from the vehicles were examined and the secondary structure of rhBMP-2 and P24 after 28 days' release process was analyzed. In vitro cell proliferation, osteogenic differentiation and rat calvarial defect repair were evaluated. The results proved that the composite vehicle had favorable compressive strength, elastic modulus, the porosity, and the bulk density. The secondary structures of rhBMP-2 and P24 kept stability during microencapsulation and release process. P24 from the vehicle kept a geared-up release and rhBMP-2 from the vehicle kept a three-stage mode release process. The results of in vitro cell detection showed that the composite vehicle had good biocompatibility and osteoinduction. In vivo rat calvarial defect repair demonstrated that both groups of vehicles with rhBMP2 and P24 exhibited satisfied bone defect repair. This research showed that the composite vehicle could exhibit sustained release of osteogenic factors. CS/nHAC/PLGA-MS loading rhBMP-2 or P24 could be a novel and ideal scaffold for bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1593-1606, 2017., (© 2017 Wiley Periodicals, Inc.)

Tissue-engineered bone (TEB) analysis in vivo relies heavily on tissue histological and end-point evaluations requiring the sacrifice of animals at specific time points. Due to differences in animal response to implanted tissues, the conventional analytical methods to evaluate TEB can introduce data inconsistencies. Additionally, the conventional methods increase the number of animals required to provide an acceptable statistical power for hypothesis testing. Alternatively, our non-invasive optical imaging allows for the longitudinal analysis of regenerating tissue, where each animal acts as its own control, thus reducing overall animal numbers. In our 6 month feasibility study, TEB, consisting of a silk protein scaffold with or without differentiated mesenchymal stem cells, was implanted in a critical-sized calvarial defect mouse model. Osteogenesis of the TEB was monitored through signal variation, using magnetic resonance imaging (MRI) and near-infrared (NIR) optical imaging with IRDye® 800CW BoneTag TM (800CW BT, a bone-specific marker used to label osteogenically differentiated mesenchymal stem cells and mineralization). Histological endpoint measurements and computed tomography (CT) were used to confirm imaging findings. Anatomical MRI revealed decreased signal intensity, indicating mineralization, in the TEB compared to the control (i.e. silk scaffold only) at various growth stages. NIR optical imaging results demonstrated a signal intensity increase of the TEB compared to control. Interpretation of the imaging results were confirmed by histological analysis. Specifically, haematoxylin and eosin staining revealing de novo bone in TEB showed that 80% of the defect was covered by TEB, while only 40% was covered for the control. Taken together, these results demonstrate the potential of multi-modal non-invasive imaging to visualize and quantify TEB for the assessment of regenerative medicine strategies. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Bone reconstruction is a challenging issue in the regeneration of surgically removed bone and disease-related bone defects. Although bone morphogenetic protein-2 (BMP-2) has received considerable attention as a bone regeneration inducer, a high dose of BMP-2 is typically required due to its short life-time under in vivo conditions. We have proposed a method to enhance the osteogenetic differentiation ability of BMP-2 in vitro that is based on supramolecular polyelectrolyte complexation with sulfonated polyrotaxanes (PRXs) consisting of sulfopropyl ether (SPE)-modified α-cyclodextrins threaded along a poly(ethylene glycol) chain capped with terminal bulky stopper molecules. In this study, we evaluated the in vivo bone regeneration ability of the SPE-PRX/BMP-2 complexes in a mouse calvarial defect model in comparison to free BMP-2 and heparin/BMP-2 complexes. The regenerated bone area was determined by X-ray computed microtomography, and the mice implanted with sulfonated PRX/BMP-2 complexes exhibited rapid and significant bone regeneration compared to those implanted with free BMP-2 and heparin/BMP-2 complexes. We concluded that the sulfonated PRX/BMP-2 complexes are a promising candidate for clinical bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1355-1363, 2017., (© 2017 Wiley Periodicals, Inc.)