3D-Printed Triply Periodic Minimal Surface Ceramic Scaffold Loaded With Bone Morphogenetic Protein-2 and Zoledronic for Cranium Defect Repairment

Critical-sized cranial bone defect remains a great clinical challenge. With advantages in regenerative medicine, injectable hydrogels incorporated with bioactive molecules show great potential in promoting cranial bone repair. Recently, we developed a dual delivery system by sequential release of bone morphogenetic protein 2 (BMP2) followed by insulin-like growth factor 1 (IGF1) in microparticles (MPs), and an injectable alginate/collagen (alg/col)-based hydrogel. In this study, we aim to evaluate the effect of dual delivery of BMP2 and IGF1 in MPs through the injectable hydrogel in critical-sized cranial bone defect healing. The gelatin MPs loaded with BMP2 and poly(lactic-co-glycolic acid)-poly(ethylene glycol)-carboxyl (PLGA-PEG-COOH) MPs loaded with IGF1 were prepared, respectively. The encapsulation efficiency and release profile of growth factors in MPs were measured. A cranial defect model was applied to evaluate the efficacy of the dual delivery system in bone regeneration. Adult Sprague Dawley rats were subjected to osteotomy to make an ⌀8-mm cranial defect. The injectable hydrogel containing MPs loaded with BMP2 (2 μg), IGF1 (2 μg), or a combination of BMP2 (1 μg) and IGF1 (1 μg) were injected to the defect site. New bone formation was evaluated by microcomputed tomography, histological analysis, and immunohistochemistry after 4 or 8 weeks. Data showed that dual delivery of the low-dose BMP2 and IGF1 in MPs through alg/col-based hydrogel successfully restored cranial bone as early as 4 weeks after implantation, whose effect was comparable to the single delivery of high-dose BMP2 in MPs. In conclusion, this study suggests that dual delivery of BMP2 and IGF1 in MPs in alg/col-based hydrogel achieves early bone regeneration in critical-sized bone defect, with advantage in reducing the dose of BMP2. Impact Statement Sequential release of bone morphogenetic protein 2 (BMP2) followed by insulin-like growth factor 1 (IGF1) in two different microparticles promotes critical-sized bone defect healing. This dual delivery system reduces the dose of BMP2 by supplementing IGF1, which may diminish the potential side effects of BMP2.

Large critical size bone defects are complicated to treat, and in many cases, autografts become a challenge due to size and availability. In such situations, a synthetic bone implant that can be patient-specifically designed and fabricated with control over parameters such as porosity, rigidity, and osteogenic cues can act as a potential synthetic bone substitute. In this study, we produced photocuring composite resins with poly(trimethylene carbonate) containing high ratios of bioactive ceramics and printed porous 3D composite scaffolds to be used as bone grafts. To enhance the overall surface area available for cell infiltration, the scaffolds were also filled with a macroporous cryogel. Furthermore, the scaffolds were functionalized with osteoactive factors: bone morphogenetic protein and zoledronic acid. The scaffolds were evaluated in vitro for biocompatibility and for functionality in vivo in critical bone defects (∼8 mm) in two clinically relevant rabbit models. These studies included a smaller study in rabbit tibia and a larger study in the rabbit cranium. It was observed that the bioactive molecule-functionalized 3D printed porous composite scaffolds provide an excellent conductive surface inducing higher bone formation and improved defect healing in both critical size long bones and cranial defects. Our findings provide strong evidence in favor of these composites as next generation synthetic bone substitutes.

Vascularization is currently considered the biggest challenge in bone tissue engineering due to necrosis in the center of large scaffolds. We established a new expendable vascular bundle model to vascularize a three-dimensional printed channeled scaffold with and without bone morphogenetic protein-2 (BMP-2) for improved healing of large segmental bone defects. Bone formation and angiogenesis in an 8 mm critical-sized bone defect in the rat femur were significantly promoted by inserting a bundle consisting of the superficial epigastric artery and vein into the central channel of a large porous polycaprolactone scaffold. Vessels were observed sprouting from the vascular bundle inserted in the central tunnel. Although the regenerated bone volume in the group receiving the scaffold and vascular bundle was similar to that of the healthy femur, the rate of union of the group was not satisfactory (25% at 8 weeks). BMP-2 delivery was found to promote not only bone formation but also angiogenesis in the critical-sized bone defects. Both insertion of the vascular bundle alone and BMP-2 loading alone induced comparable levels of angiogenesis and when used in combination, significantly greater vascular volume was observed. These findings suggest a promising new modality of treatment in large bone defects. Level of Evidence: Therapeutic level I. Impact statement Vascularization is currently the main challenge in bone tissue engineering. The combination of a vascular bundle and an osteoinductive three-dimensional printed graft significantly improved and accelerated bone regeneration and angiogenesis in critical-sized large bone defects, suggesting a promising new modality of treatment in large bone defects.

Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.

Critical-sized bone defects or CSDs result from bone loss due to trauma, tumor removal, congenital defects, or degenerative diseases. Though autologous bone transplantation is the current gold standard in treating CSDs, its limitations include donor-site morbidity, unavailability of donor bone tissues, risk of infection, and mismatch between the bone geometry and the defect site. Customized scaffolds fabricated using 3D printing and biocompatible materials can provide mechanical integrity and facilitate osseointegration. Ti-6Al-4V (Ti64) is one of the most widely used commercial alloys in orthopedics. To avoid elastic modulus mismatch between bones and Ti64, it is imperative to use porous lattice structures. Ti64 scaffolds with diamond, cubic, and triply periodic minimal surface (TPMS) gyroid lattice architectures were fabricated using selective laser melting (SLM)with pore sizes ranging from 300 to 900 μm using selective laser melting and evaluated for mechanical and biological performance. Increasing pore size led to higher porosity (up to 90.54%) and reduced mechanical properties. Young’s modulus ranged from 13.18 GPa to 1.01 GPa, while yield stress decreased from 478.16 MPa to 14.86 MPa. Diamond and cubic scaffolds with 300–600 μm pores exhibited stiffness within the cortical bone range, while the 900 μm diamond scaffold approached trabecular stiffness. Gyroid scaffolds (600–900 μm) also showed modulus and yield strength within the cortical bone range but were not suitable for trabecular applications due to their higher stiffness. Cytocompatibility was confirmed through leachate analysis and DAPI-stained osteoblast nuclei. The biological evaluation reported maximum cell adherence in lower pore sizes, with gyroid scaffolds showing a statistically significant (p < 0.01) increase in cell proliferation. These findings suggest that 300–600 μm lattice scaffolds offer an optimal balance between mechanical integrity and biological response for load-bearing bone repair.

Objective To repair a 1.5 cm critical-sized rabbit bone defect model by a novel bone bioengineering construct after co-transfection of recombinant human bone morphogenetic protein-2 （rhBMP-2） and basic fibroblast growth factor （bFGF） into bone marrow stem cells （BMSCs） and combination with nano-hydroxyapatite/recombinant human-like collagen/polylactic acid （nHA/RHLC/PLA） scaffold. Methods A 1.5 cm critical-sized bone defect model was created. BMSCs after gene transfeetion were combined with nHA/RHLC/PLA scaffolds to fabricate various biocomposites. Six groups were established： rhBMP-2- bFGF/BMSCs/nHA/RHLC/PLA （A）, rhBMP-2/BMSCs/nHA/RHLC/PLA （B）, bFGF/BMSCs/nHA/ RHLC/PLA （C） , BMSCs/nHA/RHLC/PLA （ D）, nHA/RHLC/PLA （E） and untreated bone defect group （F）. At 6th and 12th week postoperation, the samples were harvested and subjected to radiographic and histologic analyses. Results At 6th week after transplantation, obvious new bone tissue was formed in group A with bone area of （47.24 ± 4. 37 ） %, significantly greater than in group B [（28.24 ± 2. 35 ） %] and group C [（ 13.46 ± 2. 27 ） %] （ P 〈 0. 05 ）. No bone formation was observed in groups D and E. Twelve weeks later, bone integrity was restored and medullary cavity recanalized in group A with bone area of （ 96.84 ± 2. 28 ） % ; bone integrity was restored but medullary cavity was not recanalized yet in group B with bone area of （ 92. 24 ± 1.75 ） % ; slightly enhanced bone tissue formation was seen in group C [（24.73 ±2. 01 ）%] and few new bone tissue was found in group D; no bone tissue was formed in group E. The therapeutic effect for bone defect in group A was superior to other groups （ P 〈 0. 05 ）, and the bone defect in groups C, D and E was not repaired. Conclusion A new bone tissue engineering biocomposite by the combination of nHA/RHLC/PLA and BMSCs after double-transfection of rhBMP-2 and bFGF can produce apparently bone tissue in orthotopic site, which provides a new view relating to tissue-engineering repair of larze bone defect in clinical area. Key words: Recombinant bone morphogenetic protein-2; Basic fibroblast growth factor; Bonemarrow stem cells; Bone defect

Treatment of large-size bone defects is difficult, and acquiring autografts may be challenging due to limited availability. A synthetic patient-specific bone substitute can be developed by using 3D printing technologies in such cases. In the present study, we have developed photocurable composite resins with poly(trimethylene carbonate) (PTMC) containing a high percentage of biodegradable bioactive strontium-substituted nanohydroxyapatite (SrHA, size 30-70 nm). These photocurable resins have then been employed to develop high-surface-area 3D-printed bone substitutes using the digital light processing (DLP) technique. To enhance the surface area of the 3D-printed substitute, cryogels alone and functionalized with bioactive components of bone morphogenetic protein (BMP) and zoledronic acid (ZA) were filled within the 3D-printed scaffold/substitute. The scaffolds were tested in vitro for biocompatibility and functionality in vivo in two therapeutically relevant rat models with large bone defects (4 mm). The porosities of 3D printed scaffolds were found to be 60.1 ± 0.9%, 72.9 ± 0.5%, and 74.3 ± 1.6% for PTMC, PTMC-HA, and PTMC-SrHA, respectively, which is in the range of cancellous bone (50-95%). The thermogravimetric analysis demonstrated the fabrication of 3D printed composites with HA and SrHA concentrations of 51.5 and 57.4 wt %, respectively, in the PTMC matrix. The tensile Young's modulus (E), compressive moduli, and wettability increased post incorporation of SrHA and HA in the PTMC matrix. In vitro and in vivo results revealed that SrHA integrated into the PTMC matrix exhibited good physicochemical and biological properties. Furthermore, the osteoactive molecule-functionalized 3D printed composite scaffolds were found to have an adequate osteoconductive and osteoinductive surface that has shown increased bone regeneration and defect repair in both tibial and cranial bone defects. Our findings thus support the use of PTMC-SrHA composites as next-generation patient-specific synthetic bioactive biodegradable bone substitutes.

The aging population has rapidly driven the demand for bone regeneration. The pore structure of a scaffold is a critical factor that affects its mechanical strength and bone regeneration. Triply periodic minimal surface gyroid structures similar to the trabecular bone structure are considered superior to strut-based lattice structures (e.g., grids) in terms of bone regeneration. However, at this stage, this is only a hypothesis and is not supported by evidence. In this study, we experimentally validated this hypothesis by comparing gyroid and grid scaffolds composed of carbonate apatite. The gyroid scaffolds possessed compressive strength approximately 1.6-fold higher than that of the grid scaffolds because the gyroid structure prevented stress concentration, whereas the grid structure could not. The porosity of gyroid scaffolds was higher than that of the grid scaffolds; however, porosity and compressive strength generally have a trade-off relationship. Moreover, the gyroid scaffolds formed more than twice the amount of bone as grid scaffolds in a critical-sized bone defect in rabbit femur condyles. This favorable bone regeneration using gyroid scaffolds was attributed to the high permeability (i.e., larger volume of macropores or porosity) and curvature profile of the gyroid structure. Thus, this study validated the conventional hypothesis using in vivo experiments and revealed factors that led to this hypothetical outcome. The findings of this study are expected to contribute to the development of scaffolds that can achieve early bone regeneration without sacrificing the mechanical strength.

Modifying the Proliferative State of Target Cells to Control DNA Expression and Identifying Cell Types Transfected In Vivo

Triply Periodic Minimal Surface (TPMS)-based aluminium–alumina Interpenetrating Phase Composites (IPCs) manufactured through the combination of Additive Manufacturing (AM) and investment casting are explored in this study. Multiple alumina TPMS structures (Gyroid, Diamond, and Primitive) with different geometries and volume fractions were designed and fabricated using Digital Light Processing (DLP) AM technology. Afterwards, these ceramic structures were filled with an aluminium alloy via investment casting, uncovering an aluminium–alumina IPCs. A global characterization was performed, including ceramics shrinkage and mass loss; specimens’ morphology; chemical and crystalline characterization; density analysis and mechanical testing. Overall, DLP technology was found effective for producing these highly complex ceramic structures, with high surface quality. The sintered alumina structures presented a relative density of ca. 76.3% and a pseudo-ductile layer-by-layer failure behaviour, with Diamond-based TPMS exhibiting the highest compressive strength. Regarding the IPCs, the addition of aluminium significantly changed the compressive behaviour of the samples, presenting an energy absorption behaviour. The integration of the alumina phase into the aluminium alloy led to an improvement on the compressive offset stress of approximately 6% when compared to the aluminium alloy used. Diamond and Gyroid IPCs demonstrated similar mechanical behaviour and the highest mechanical performance.Graphical

ObjectiveTo investigate the therapeutic efficacy of total flavonoids of Rhizoma Drynariae (TFRD) in conjunction with a calcium phosphate/collagen scaffold for the repair of cranial defects in rats.MethodsThe subjects, rats, were segregated into four groups: Control, TFRD, Scaffold, and TFRD + Scaffold. Cranial critical bone defects, 5 mm in diameter, were artificially induced through precise drilling. Post-surgery, at intervals of 2, 4, and 8 weeks, micro-CT scans were conducted to evaluate the progress of skull repair. Hematoxylin–eosin and Masson staining techniques were applied to discern morphological disparities, and immunohistochemical staining was utilized to ascertain the expression levels of local osteogenic active factors, such as bone morphogenetic protein 2 (BMP-2) and osteocalcin (OCN).ResultsUpon examination at the 8-week mark, cranial defects in the Scaffold and TFRD + Scaffold cohorts manifested significant repair, with the latter group displaying only negligible foramina. Micro-CT examination unveiled relative to its counterparts, and the TFRD + Scaffold groups exhibited marked bone regeneration at the 4- and 8-week intervals. Notably, the TFRD + Scaffold group exhibited substantial bone defect repair compared to the TFRD and Scaffold groups throughout the entire observation period, while histomorphological assessment demonstrated a significantly higher collagen fiber content than the other groups after 2 weeks. Immunohistochemical analysis further substantiated that the TFRD + Scaffold had augmented expression of BMP-2 at 2, 4 weeks and OCN at 2 weeks relative to other groups.ConclusionsThe synergistic application of TFRD and calcium phosphate/collagen scaffold has been shown to enhance bone mineralization, bone plasticity, and bone histomorphology especially during initial osteogenesis phases.

Preparation and properties of K0.48Na0.52NbO3 ceramics for bone scaffolds via digital light processing

In injection molding, cooling channels are usually manufactured with a straight shape, and thus have low cooling efficiency for a curved mold. Recently, additive manufacturing (AM) was used to fabricate conformal cooling channels that could maintain a consistent distance from the curved surface of the mold. Because this conformal cooling channel was designed to obtain a uniform temperature on the mold surface, it could not efficiently cool locally heated regions (hot spots). This study developed an adaptive conformal cooling method that supports localized-yet-uniform cooling for the heated region by employing micro-cellular cooling structures instead of the typical cooling channels. An injection molding simulation was conducted to predict the locally heated region, and a mold core was designed to include a triply periodic minimal surface (TPMS) structure near the heated region. Two biomimetic TPMS structures, Schwarz-diamond and gyroid structures, were designed and fabricated using a digital light processing (DLP)-type polymer AM process. Various design parameters of the TPMS structures, the TPMS shapes and base coordinates, were investigated in terms of the conformal cooling performance. The mold core with the best TPMS design was fabricated using a powder-bed fusion (PBF)-type metal AM process, and injection molding experiments were conducted using the additively manufactured mold core. The developed mold with TPMS cooling achieved a 15 s cooling time to satisfy the dimensional tolerance, which corresponds to a 40% reduction in comparison with that of the conventional cooling (25 s).

The aim of this study was to investigate the in vitro osteogenic capacity of bone morphogenetic protein 7 (BMP-7) overexpressing adipose-derived (Ad-) mesenchymal stem cells (MSCs) sheets (BMP-7-CS). In addition, BMP-7-CS were transplanted into critical-sized bone defects and osteogenesis was assessed. BMP-7 gene expressing lentivirus particles were transduced into Ad-MSCs. BMP-7, at the mRNA and protein level, was up-regulated in BMP-7-MSCs compared to expression in Ad-MSCs. Osteogenic and vascular-related gene expressions were up-regulated in BMP-7-CS compared to Ad-MSCs and Ad-MSC sheets. In a segmental bone-defect model, newly formed bone and neovascularization were enhanced with BMP-7-CS, or with a combination of BMP-7-CS and demineralized bone matrix (DBM), compared to those in control groups. These results demonstrate that lentiviral-mediated gene transfer of BMP-7 into Ad-MSCs allows for stable BMP-7 production. BMP-7-CS displayed higher osteogenic capacity than Ad-MSCs and Ad-MSC sheets. In addition, BMP-7-CS combined with demineralized bone matrix (DBM) stimulated new bone and blood vessel formation in a canine critical-sized bone defect. The BMP-7-CS not only provides BMP-7 producing MSCs but also produce osteogenic and vascular trophic factors. Thus, BMP-7-CS and DBM have therapeutic potential for the treatment of critical-sized bone defects and could be used to further enhance clinical outcomes during bone-defect treatment.

ObjectiveTo compare new bone formation in mandibular symphysis critical-sized bone defects (CSBDs) in healthy and osteoporotic rats filled with bioceramics (BCs) with or without buccal fat pad mesenchymal stem cells (BFPSCs).Materials and methodsThirty-two adult female Sprague–Dawley rats were randomized to two groups (n = 16 per group): group 1 healthy and group 2 osteoporotic (with bilateral ovariectomy). The central portion of the rat mandibular symphysis was used as a physiological CSBD. In each group, eight defects were filled with BC (hydroxyapatite 60% and β-tricalcium phosphate 40%) alone and eight with BFPSCs cultured on BC. The animals were sacrificed at 4 and 8 weeks, and the mandibles were processed for micro-computed tomography to analyze radiological union and bone mineral density (BMD); histological analysis of the bone union; and immunohistochemical analysis, which included immunoreactivity of vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP-2).ResultsIn both groups, CSBDs filled with BC + BFPSCs showed greater radiological bone union, BMD and histological bone union, and more VEGF and BMP-2 positivity, compared with CSBDs treated with BC alone at 4 and 8 weeks.ConclusionsThe application of BFPSCs cultured on BCs improves bone regeneration in CSBDs compared with BCs alone in healthy and osteoporotic rats.Clinical relevanceOur results may aid bone regeneration of maxillofacial CSBDs of both healthy and osteoporotic patients, but further studies are necessary.