Critical-sized Rat Calvarial Defects Research Articles

Octacalcium phosphate crystals including a higher density dislocation improve its materials osteogenecity

Herein, we show that the enhanced osteogenecity of octacalcium phosphate (OCP) biomaterial, recently identified as an important element in hybrid organic–inorganic nanocomposites involved in the initial hydroxyapatite crystal expansion in mammal bones, results from an enhanced chemical property, stemming from the presence of lattice strain and dislocations. Two types of OCPs were synthesized by wet-chemical processing in the presence (c-OCP) and absence (w-OCP) of gelatin, respectively, and subjected to structural, chemical, and biological analyses. High-resolution transmission electron microscopy (HRTEM) and fast Fourier transform (FFT) analyses revealed that c-OCP crystals contained approximately six times higher edge dislocations with Burgers vectors perpendicular to a-axis than that in the case of w-OCP. The dissolution of c-OCP crystal in tris-HCl buffer occurred toward the long axis of the crystal, most likely, toward the lattice strain along the c-axis direction, while w-OCP crystal dissolved toward the a-axis direction. The study suggested that the increment of internal energy by the higher dislocation density contributed promoting c-OCP dissolution and hydrolysis through decreasing the activation energy. c-OCP crystal displayed enhanced in vitro mesenchymal stem 2D cell and 3D spheroid differentiation, in vivo bone formation, and apatite crystallographic orientation in critical-sized rat calvarial defect model as compared to w-OCP crystal, at the same time, converting to apatite structure earlier than w-OCP. The present study demonstrates that dislocation-related dissolution along with enhanced conversion of OCP is a determinant in bone induction, which may be relevant to normal bone development utilizing OCP biomaterials.

Exogenous adenosine activates A2A adenosine receptor to inhibit RANKL-induced osteoclastogenesis via AP-1 pathway to facilitate bone repair

Adenosine is a purine nucleoside involved in regulating bone homeostasis through binding to A1, A2A, A2B, and A3 adenosine receptors (A1R, A2AR, A2BR, and A3R, respectively). However, the underlying mechanisms by which adenosine and receptor subtypes regulate osteoclast differentiation remain uncertain. This study aims to assess the role of exogenous adenosine and receptor subtypes in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation and explore the underlying molecular mechanisms. The nanofibrous mats incorporated with adenosine exhibited robust ability to facilitate rat critical-size calvarial defect healing with decreased number of osteoclasts. Moreover, exogenous adenosine substantially enhanced the expression of A2AR and suppressed tartrate-resistant acid phosphatase-positive osteoclast formation and expression of osteoclast-related genes Ctsk, NFATc1, MMP9, and ACP5. This enhancement and suppression could be reversed by adding an A2AR antagonist, ZM241385, in RAW264.7 cells. Finally, RNA sequencing showed that the expression of Fos-related antigen 2 (Fra2) was distinctly downregulated through stimulation of adenosine in RAW264.7 cells treated with RANKL. This downregulation was reversed by ZM241385 according to real-time PCR, Western blot, and immunofluorescence analyses. These findings demonstrated that exogenous adenosine binding to A2AR attenuated osteoclast differentiation via the inhibition of activating protein-1 (AP-1, including Fra2 subunit) pathway both in vitro and in vivo.

PLGA/β-TCP composite scaffold incorporating cucurbitacin B promotes bone regeneration by inducing angiogenesis.

ObjectivesVascularization is an essential step in successful bone tissue engineering. The induction of angiogenesis in bone tissue engineering can be enhanced through the delivery of therapeutic agents that stimulate vessel and bone formation. In this study, we show that cucurbitacin B (CuB), a tetracyclic terpene derived from Cucurbitaceae family plants, facilitates the induction of angiogenesis in vitro.MethodsWe incorporated CuB into a biodegradable poly (lactide-co-glycolide) (PLGA) and β-tricalcium phosphate (β-TCP) biomaterial scaffold (PT/CuB) Using 3D low-temperature rapid prototyping (LT-RP) technology. A rat skull defect model was used to verify whether the drug-incorporated scaffold has the effects of angiogenesis and osteogenesis in vivo for the regeneration of bone defect. Cytotoxicity assay was performed to determine the safe dose range of the CuB. Tube formation assay and western blot assay were used to analyze the angiogenesis effect of CuB.ResultsPT/CuB scaffold possessed well-designed bio-mimic structure and improved mechanical properties. CuB was linear release from the composite scaffold without affecting pH value. The results demonstrated that the PT/CuB scaffold significantly enhanced neovascularization and bone regeneration in a rat critical size calvarial defect model compared to the scaffold implants without CuB. Furthermore, CuB stimulated angiogenic signaling via up-regulating VEGFR2 and VEGFR-related signaling pathways.ConclusionCuB can serve as promising candidate compound for promoting neovascularization and osteogenesis, especially in tissue engineering for repair of bone defects.The translational potential of this articleThis study highlights the potential use of CuB as a therapeutic agent and strongly support its adoption as a component of composite scaffolds for tissue-engineering of bone repair.

Self-assembled nanocomposite hydrogels enhanced by nanoparticles phosphonate-magnesium coordination for bone regeneration

The repair of critical-sized bone defects remains challenging. Natural polysaccharide-based hydrogels (NPHs) have shown great promise for facilitating bone defect healing, but limited by osteogenesis, mechanical strength and stability. Here we prepared a water-soluble methacrylic anhydride-co-phosphate modified chitosan (CSMAP) incorporated MgO nanoparticles to form a pergel, and followed by introduction of acrylamide to construct a double-network hydrogel (CSMAP-MgO). We found that Mg2+ released from CSMAP-MgO hydrogel in a stable and sustained manner in 30 days and the compressive strength was maintained and the compressive modulus was even enhanced during immersion in PBS for 28 days. Moreover, the CSMAP-MgO hydrogel was superior in promoting in vitro calcium phosphate deposition, MC3T3-E1 cell proliferation, osteogenic differentiation, and mineralization compared to Magnesium free groups. At 12 weeks post-implantation into a rat critical-sized calvarial defect, the CSMAP-MgO hydrogel promoted significantly more new bone formation compared to CSMAP and non-treated groups. Thus, the CSMAP-MgO hydrogel has great promising as a bone filling material.

Rat Calvarial Bone Regeneration by 3D-Printed β-Tricalcium Phosphate Incorporating MicroRNA-200c.

Advanced fabrication methods for bone grafts designed to match defect sites that combine biodegradable, osteoconductive materials with potent, osteoinductive biologics would significantly impact the clinical treatment of large bone defects. In this study, we engineered synthetic bone grafts using a hybrid approach that combined three-dimensional (3D-)printed biodegradable, osteoconductive β-tricalcium phosphate (β-TCP) with osteoinductive microRNA(miR)-200c. 3D-printed β-TCP scaffolds were fabricated utilizing a suspension-enclosing projection-stereolithography (SEPS) process to produce constructs with reproducible microarchitectures that enhanced the osteoconductive properties of β-TCP. Collagen coating on 3D-printed β-TCP scaffolds slowed the release of plasmid DNA encoding miR-200c compared to noncoated constructs. 3D-printed β-TCP scaffolds coated with miR-200c-incorporated collagen increased the transfection efficiency of miR-200c of both rat and human BMSCs and additionally increased osteogenic differentiation of hBMSCs in vitro. Furthermore, miR-200c-incorporated scaffolds significantly enhanced bone regeneration in critical-sized rat calvarial defects. These results strongly indicate that bone grafts combining SEPS 3D-printed osteoconductive biomaterial-based scaffolds with osteoinductive miR-200c can be used as superior bone substitutes for the clinical treatment of large bone defects.

Biodegradable dual-crosslinked adhesive glue for fixation and promotion of osteogenesis

The effective fixation of fracture fragments is the foundation for the smooth healing process of fracture. Due to the presence of small fracture fragments, the comminuted fracture cannot be fixed under high mechanical strength leading to the bone nonunion and delayed healing. In this study, the primary crosslinked injectable bone glue was fabricated by the Schiff base reaction between low substitution degree methacrylate gelatin (GelMa) and Oxidized dextran (oDex) covalently crosslinked with Aminated mesoporous bioactive glass nanoparticles (AMBGN), then the second crosslinked bone glue was formed among small fracture fragments after the irradiation. By this method, the osteogenic dual-crosslinking biodegradable bone glue was synthesized with enhanced mechanical capability. This bone glue was biocompatible and biodegradable, in addition, after the second crosslinking, the mechanical strength was 2.1 times higher than that in primary cross-linking. Moreover, the dual-crosslinked bone glue also exhibited excellent adhesion ability on the isolated rat bone tissues. Additionally, the bone glue can increase the proliferation of bone marrow mesenchymal stem cells (BMSCs) and enhance the expression of Alkaline phosphatase (ALP) and the deposition of calcium nodules with the up-regulating expression of the osteogenic protein (Runx2) and adhesin (Vinculin) in vitro. Furthermore, the bone glue also presented enhanced osteogenesis in the rat calvarial critical-size defect models in vivo. Overall, this osteogenic dual-crosslinking biodegradable bone glue with high mechanical strength provided a new approach in treating the comminuted fracture fragments.

Involvement of distant octacalcium phosphate scaffolds in enhancing early differentiation of osteocytes during bone regeneration

This study hypothesized that distant octacalcium phosphate (OCP) scaffolds may enhance osteocyte differentiation in newly formed bone matrices. The results obtained were compared with those of Ca-deficient hydroxyapatite (OCP hydrolyzate, referred to as HL hereafter). Granular OCP and HL, 300–500 µm in diameter, were implanted in critical-sized rat calvarial defects for eight weeks and subjected to histology, immunohistochemistry, histomorphometry, and transmission electron microscopy (TEM). Early osteocyte differentiation from an osteoblastic cell line (IDG-SW3) was examined using materials without contacting the surfaces for 10 days. The material properties and the medium composition were analyzed through selected area electron diffraction (SAED) using TEM observation and curve fitting of Fourier transform infrared (FT-IR) spectroscopy. The number of positive cells of an osteocyte earlier differentiation marker podoplanin (PDPN) in bone matrices, along the direction of bone formation, was significantly higher in OCP than that in HL. The ultrastructure around the OCP surfaces observed by TEM showed the infiltration of some cells, including osteocytes adjacent to the OCP surface layers. The OCP structure remained unchanged by SAED analysis. Nanoparticle deposition and hydrolysis on OCP surfaces were detected by TEM and FT-IR, respectively, during early osteocyte differentiation in vitro. The medium saturation degree varied in accord with ionic dissolution, resulting in possible hydroxyapatite formation on OCP but not on HL. These results suggested that OCP stimulates early osteocyte differentiation in the bone matrix from a distance through its metastable chemical properties. Statement of significanceThis study demonstrated that octacalcium phosphate (OCP) implanted in critical-sized rat calvaria bone defects is capable of enhancing the early differentiation of osteocytes embedded in newly formed bone matrices, even when the surface OCP is separated from the osteocytes. This prominent bioactive property of OCP was demonstrated by comparing the in vivo and in vitro performances with a control material, Ca-deficient hydroxyapatite (OCP hydrolyzate). The findings were elucidated by histomorphometry, which analyzed the differentiation of osteocytes along the parallel direction of new bone growth by osteoblasts. Therefore, OCP should stimulate osteocyte differentiation through ionic dissolution even in vivo owing to its metastable chemical properties, as previously reported in an in vitro study (Acta Biomater 69:362, 2018).

Polyelectrolyte multilayer composite coating on 316 L stainless steel for controlled release of dual growth factors accelerating restoration of bone defects

A composite coating of polyelectrolyte multilayers (PEMs) consisting of collagen, a chitosan barrier, and poly-γ-glutamic acid was fabricated using a spin coating technique to investigate and overcome the limited osseointegration capacity of 316 L stainless steel (316 L SS). To further enhance the biocompatibility, bone morphogenetic protein 2 (BMP-2) and basic fibroblast growth factor-2 (FGF-2) were loaded separately as dual growth factors, allowing for progressive drug release following the natural process of bone regeneration. The first burst release of FGF-2 triggered the proliferation of surrounding cells, and the subsequent release of BMP-2 stimulated their differentiation. The microstructure, surface potential, hardness, reduced Young's modulus, and wettability were assessed using scanning electron microscopy, nanoindentation, and water contact angle. The formation of apatite layers after immersion in simulated body fluid confirmed the bioactivity of this PEM. PEMs loaded with BMP-2 and FGF-2 showed a long sustained release of growth factors for up to 48 days. The biological properties were studied in vitro with rat bone mesenchymal stem cells (rBMSCs) and in vivo using a rat critical-sized calvarial defect model. PEMs loaded with growth factors further stimulated the proliferation and osteogenic differentiation of rBMSCs and the histology results indicated that new bone tissues could directly grow onto the PEMs. These findings suggest that PEM composite coating possesses significant potential for surface modification and long-term drug release of metallic implants to assist with bone restoration.

Effect of wheat gluten on improved thermal cross-linking and osteogenesis of hydroxyapatite-gelatin composite scaffolds

Promising strategies to stabilize gelatin or collagen include glutaraldehyde-based chemical cross-linking or dehydrothermal treatment at different temperatures (120–180 °C). However, these procedures require 24–48 h for complete cross-linking to occur. The present study aims to evaluate the role of wheat gluten on enhancing thermal cross-linking of silica-nanohydroxyapatite (nanoHA)-gelatin composite scaffolds within a shorter period (2 h). Changes in properties were evaluated by varying the ratio of gelatin and gluten in silica-nanoHA matrix (60 wt% ceramic: 40 wt% polymer). The results showed that the scaffolds cross-linked at 170 °C were stable in phosphate-buffered saline for 21 days. It was crystalline and porous in nature. However, the scaffolds with high weight percentage of wheat gluten were brittle, while those with low gluten degraded fast in vitro. The mesenchymal stem cells could adhere, proliferate and differentiate into osteogenic lineage on wheat gluten-containing scaffolds for 21 days (mainly medium concentration). The scaffold also supported new bone formation in critical-sized rat calvarial defect, showing its osteoconductive and osteointegrative nature. In short, this study showed the potential of wheat gluten on improving thermal cross-linking within a shorter period and its suitability to use as a biomimetic bone graft for bone tissue engineering.

In situ bone regeneration with sequential delivery of aptamer and BMP2 from an ECM-based scaffold fabricated by cryogenic free-form extrusion

In situ tissue engineering is a powerful strategy for the treatment of bone defects. It could overcome the limitations of traditional bone tissue engineering, which typically involves extensive cell expansion steps, low cell survival rates upon transplantation, and a risk of immuno-rejection. Here, a porous scaffold polycaprolactone (PCL)/decellularized small intestine submucosa (SIS) was fabricated via cryogenic free-form extrusion, followed by surface modification with aptamer and PlGF-2123-144*-fused BMP2 (pBMP2). The two bioactive molecules were delivered sequentially. The aptamer Apt19s, which exhibited binding affinity to bone marrow-derived mesenchymal stem cells (BMSCs), was quickly released, facilitating the mobilization and recruitment of host BMSCs. BMP2 fused with a PlGF-2123-144 peptide, which showed “super-affinity” to the ECM matrix, was released in a slow and sustained manner, inducing BMSC osteogenic differentiation. In vitro results showed that the sequential release of PCL/SIS-pBMP2-Apt19s promoted cell migration, proliferation, alkaline phosphatase activity, and mRNA expression of osteogenesis-related genes. The in vivo results demonstrated that the sequential release system of PCL/SIS-pBMP2-Apt19s evidently increased bone formation in rat calvarial critical-sized defects compared to the sequential release system of PCL/SIS-BMP2-Apt19s. Thus, the novel delivery system shows potential as an ideal alternative for achieving cell-free scaffold-based bone regeneration in situ.

Spongostan™ Leads to Increased Regeneration of a Rat Calvarial Critical Size Defect Compared to NanoBone® and Actifuse.

Bone substitute materials are becoming increasingly important in oral and maxillofacial surgery. Reconstruction of critical size bone defects is still challenging for surgeons. Here, we compared the clinically applied organic bone substitute materials NanoBone® (nanocrystalline hydroxyapatite and nanostructured silica gel; n = 5) and Actifuse (calcium phosphate with silicate substitution; n = 5) with natural collagen-based Spongostan™ (hardened pork gelatin containing formalin and lauryl alcohol; n = 5) in bilateral rat critical-size defects (5 mm diameter). On topological level, NanoBone is known to harbour nanopores of about 20 nm diameter, while Actifuse comprises micropores of 200–500 µm. Spongostan™, which is clinically applied as a haemostatic agent, combines in its wet form both nano- and microporous topological features by comprising 60.66 ± 24.48 μm micropores accompanied by nanopores of 32.97 ± 1.41 nm diameter. Micro-computed tomography (µCT) used for evaluation 30 days after surgery revealed a significant increase in bone volume by all three bone substitute materials in comparison to the untreated controls. Clearly visual was the closure of trepanation in all treated groups, but granular appearance of NanoBone® and Actifuse with less closure at the margins of the burr holes. In contrast, transplantion of Spongostan™ lead to complete filling of the burr hole with the highest bone volume of 7.98 ccm and the highest bone mineral density compared to all other groups. In summary, transplantation of Spongostan™ resulted in increased regeneration of a rat calvarial critical size defect compared to NanoBone and Actifuse, suggesting the distinct nano- and microtopography of wet Spongostan™ to account for this superior regenerative capacity. Since Spongostan™ is a clinically approved product used primarily for haemostasis, it may represent an interesting alternative in the reconstruction of defects in the maxillary region.

Simvastatin loaded chitosan guided bone regeneration membranes stimulate bone healing.

Electrospun chitosan membranes (ESCM) modified with short-chain fatty acids have the ability to control the release of simvastatin (SMV), an anti-cholesterol drug with osteogenic potential, for guided bone regeneration (GBR) applications. This study evaluated in vivo osteogenic effects of rapid short release of SMV (4weeks) vs long sustained release (8weeks) from acetic anhydride (AA)-and hexanoic anhydride (HA)-modified ESCMs, respectively. AA ESCMs loaded with 10 or 50µg SMV and HA ESCMs loaded with 50µg SMV were evaluated for biocompatibility and bone formation at 4 and 8weeks, in 5mm critical size rat calvarial defects, using histological evaluation and micro-CT analysis. No severe inflammatory response was noticed around the ESCMs. Less hydrophobic AA membranes showed signs of resorption by week 4 and were almost completely resorbed by week 8 whereas the more hydrophobic HA membranes resorbed slowly, remaining intact over 8weeks. In micro-CT analysis, 10µg SMV-loaded AA membranes did not show significant bone formation as compared to non-loaded AA membranes at either evaluation time points. 50µg SMV-loaded AA membranes stimulated significantly more bone formation than non-loaded AA membranes by week 4 (%bone=31.0±5.9% (AA50) vs 18.5±13.7% (AA0)) but showed no difference at week 8. HA membranes with 50µg SMV showed significantly more bone formation as compared to corresponding non-loaded membranes by week 8 (%bone=61.7±8.9% (HA50) vs 33.9±29.7% (HA0)), though such an effect was not significant at week 4. These results indicate that modified ESCMs may be used to control the release of SMV and promote bone healing in GBR applications.

Xenogeneic bone mineral is efficient for the repair of critical-sized rat calvarial defects

Aim. To evaluate the efficiency of bone repair on a critical-sized rat calvarial defect model using our original xenogeneic bone mineral, widely established Geistlich Bio-Oss®, and autologous bone graft.Materials and Methods. We created a critical-sized calvarial defect in Sprague-Dawley rats (n = 48) and then divided them into 4 groups (unfilled defect, autologous bone graft, Geistlich BioOss® and our original xenogeneic bone mineral, 12 rats per group). Rats were sacrificed upon 4 and 12 months (6 rats per time point) with the following excision of the implant and adjacent tissues. 3D structure, extent of mineralisation, and bone volume were measured by means of microcomputed tomography. Microanatomy of the explants and adjacent tissue was investigated by haematoxylin and eosin staining.Results. The highest and the lowest bone volume was expectedly detected when the defect was filled with the autologous bone graft or remained unfilled, respectively. Replacement of the defect by the original bone mineral entailed better regeneration as compared to Geistlich Bio-Oss. Bone mineral density, bone thickness and the extent of mineralisation did not differ significantly between the experimental groups and were close to the positive control values, indicating efficient bone repair.Conclusions. Original xenogeneic bone mineral promotes induction of bone regeneration as compared to Geistlich Bio-Oss®, a commercially available bone mineral widely used in the clinical practice.

Prevascularized hydrogels with mature vascular networks promote the regeneration of critical-size calvarial bone defects in vivo.

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 (8mm), 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.

Construction of a nanofiber network within 3D printed scaffolds for vascularized bone regeneration.

Three-dimensional (3D) printed scaffolds provide a promising prospective for application in bone tissue engineering. 3D printed scaffolds with micro- and nano-fibrous structures that facilitate cell adhesion and migration, and combined vascularization and osteoinduction bioactivity will be ideal implants for bone defect repair. Here, we fabricated a 3D printed biodegradable poly (glycerol-co-sebacic acid-co-l-lactic acid-co-polyethylene glycol) (PGSLP)-based scaffold that was internally filled with gelatin nanofibers and allowed the local release of deferoxamine (DFO), which is essential for angiogenesis and osteogenesis in bone regeneration. The nanofibrous structured gelatin/PGSLP (NGP) scaffold was fabricated using a thermally induced phase separation (TIPS) technique, and the macroporous structured gelatin/PGSLP (MGP) scaffold was prepared by directly freeze-drying. The in vitro experiments demonstrated that both DFO-loaded NGP and DFO-loaded MGP scaffolds can promote the migration and tubular formation of human umbilical vein endothelial cells (HUVECs), and enhance the mineralized nodule formation and osteogenic-related gene expression during osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). In a rat critical-sized calvarial defect model, the results suggested that the scaffolds with DFO loading significantly promote the vascular formation and accelerate bone regeneration, while the enhancement of vascularization and osteogenesis in vivo in DFO-loaded NGP scaffold was better than that in DFO-loaded MGP scaffold. Therefore, the constructed PGLSP-based scaffolds with micro- and nano-fibrous structures would be promising candidates to match the structural and functional requirements for vascularized bone regeneration.

Layered Antimicrobial Selenium Nanoparticle–Calcium Phosphate Coating on 3D Printed Scaffolds Enhanced Bone Formation in Critical Size Defects

Preventing bacterial colonization on scaffolds while supporting tissue formation is highly desirable in tissue engineering as bacterial infection remains a clinically significant risk to any implanted biomaterials. Elemental selenium (Se0) nanoparticles have emerged as a promising antimicrobial biomaterial without tissue cell toxicity, yet it remains unknown if their biological properties are from soluble Se ions or from direct cell-nanoparticle interactions. To answer this question, in this study, we developed a layered coating consisting of a Se nanoparticle layer underneath a micrometer-thick, biomimetic calcium phosphate (CaP) layer. We showed, for the first time, that the release of soluble HSe- ions from the Se nanoparticles strongly inhibited planktonic growth and biofilm formation of key bacteria, Staphylococcus aureus. The Se-CaP coating was found to support higher bone formation than the CaP-only coating in critical-size calvarial defects in rats; this finding could be directly attributed to the released soluble Se ions as the CaP layers in both groups had no detectable differences in the porous morphology, chemistry, and release of Ca or P. The Se-CaP coating was highly versatile and applicable to various surface chemistries as it formed through simple precipitation from aqueous solutions at room temperature and therefore could be promising in bone regeneration scaffolds or orthopedic implant applications.

Accelerating bone healing in vivo by harnessing the age-altered activation of c-Jun N-terminal kinase 3

We have recently demonstrated that c-Jun N-terminal kinase 3 (JNK3) is a key modulator of the enhanced osteogenic potential of stem cells derived from children when compared to those derived from adults. In this study, we formulated a JNK3-activator nanoparticle (JNK3*) that recapitulates the immense osteogenic potential of juvenile cells in adult stem cells by facilitating JNK3 activation. Moreover, we aimed to functionalize a collagen-based scaffold by incorporating the JNK3* in order to develop an advanced platform capable of accelerating bone healing by recruitment of host stem cells. Our data, in vitro and in vivo, demonstrated that the immense osteogenic potential of juvenile cells could be recapitulated in adult stem cells by facilitating JNK3 activation. Moreover, our results revealed that the JNK3* functionalized 3D scaffold induced the fastest bone healing and greatest blood vessel infiltration when implanted in critical-size rat calvarial defects in vivo. JNK3*scaffold fastest bone healing in vivo was associated with its capacity to recruit host stem cells to the site of injury and promote angiogenic-osteogenic coupling (e.g. Vegfa, Tie1, Runx2, Alp and Igf2 upregulation). In summary, this study has demonstrated the potential of harnessing knowledge of age-altered stem cell mechanobiology in order to develop a materials-based functionalization approach for the repair of large tissue defects.

Mineralized Human Amniotic Membrane as a Biomimetic Scaffold for Hard Tissue Engineering Applications.

The human amniotic membrane (HAM) has been viewed as a potential regenerative material for a wide variety of injured tissues because of its collagen-rich content. High degradability of HAM limits its wide practical application in bone tissue engineering. In this study, the natural matrix of the decellularized amniotic membrane was developed by the double diffusion method. The results confirmed a reduction of the amniotic membrane's degradability because of the deposition of calcium and phosphate ions during the double diffusion process. Real-time PCR results showed a high expression of osteogenesis-related genes from adipose-derived mesenchymal stem cells (ADMSCs) cultured on the surface of the developed mineralized amniotic membrane (MAM). Further in vivo experiments were conducted using an MAM preseeded with ADMSCs and a critical-size rat calvarial defect model. Histopathological results confirmed that the MAM + cell sample has excellent potential in bone regeneration.

Rat Calvarial Bone Regeneration by 3D-Printed Beta-Tricalcium Phosphate Incorporating MicroRNA-200c

Advanced fabrication methods of bone grafts designed to match defect sites that combine biodegradable, osteoconductive materials with potent, osteoinductive biologics would significantly impact the clinical treatment of large bone defects. In this study, we engineered synthetic bone grafts using a hybrid material approach that combined 3D-printed biodegradable, osteoconductive β-tricalcium phosphate (β-TCP) with osteoinductive microRNA(miR)-200c. 3D-printed β-TCP scaffolds were fabricated utilizing suspension-enclosing projection-stereolithography (SEPS) process to produce constructs with reproducible microarchitectures that enhanced the osteoconductive properties of β-TCP. 3D-printed β-TCP scaffolds coated with miR-200c incorporated collagen increased the transfection efficiency of miR-200c and osteogenic differentiation of hBMSCs in vitro. Furthermore, these hybrid material, miR-incorporated scaffolds significantly enhanced bone regeneration in critical-sized rat calvarial defects. These results strongly indicate that through use of SEPS 3D-printing technology, hybrid scaffolds combined by osteoconductive biomaterials and osteoinductive biologics can be used as superior bone substitutes for the clinical treatment of large bone defects.

Effects of electromagnetic fields treatment on rat critical-sized calvarial defects with a 3D-printed composite scaffold

BackgroundCurrent strategies for craniofacial defect are faced with unmet outcome. Combining 3D-printing with safe, noninvasive magnetic therapy could be a promising breakthrough.MethodsIn this study, polylactic acid/hydroxyapatite (PLA/HA) composite scaffold was fabricated. After seeding rat bone marrow mesenchymal stem cells (BMSCs) on scaffolds, the effects of electromagnetic fields (EMF) on the proliferation and osteogenic differentiation capacity of BMSCs were investigated. Additionally, 6-mm critical-sized calvarial defect was created in rats. BMSC-laden scaffolds were implanted into the defects with or without EMF treatment.ResultsOur results showed that PLA/HA composite scaffolds exhibited uniform porous structure, high porosity (~ 70%), suitable compression strength (31.18 ± 4.86 MPa), modulus of elasticity (10.12 ± 1.24 GPa), and excellent cyto-compatibility. The proliferation and osteogenic differentiation capacity of BMSCs cultured on the scaffolds were enhanced with EMF treatment. Mechanistically, EMF exposure functioned partly by activating mitogen-activated protein kinase (MAPK) or MAPK-associated ERK and JNK pathways. In vivo, significantly higher new bone formation and vascularization were observed in groups involving scaffold, BMSCs, and EMF treatment, compared to scaffold alone. Furthermore, after 12 weeks of implanting, craniums in groups including scaffold, BMSCs, and EMF exposure showed the greatest biomechanical properties.ConclusionIn conclusion, EMF treatment combined with 3D-printed scaffold has great potential applications in craniofacial regeneration.