BMP-2-immobilized PCL 3D printing scaffold with a leaf-stacked structure as a physically and biologically activated bone graft.

Although three-dimensional (3D) printing techniques are used to mimic macro- and micro-structures as well as multi-structural human tissues in tissue engineering, efficient target tissue regeneration requires bioactive 3D printing scaffolds. In this study, we developed a bone morphogenetic protein-2 (BMP-2)-immobilized polycaprolactone (PCL) 3D printing scaffold with leaf-stacked structure (LSS) (3D-PLSS-BMP) as a bioactive patient-tailored bone graft. The unique LSS was introduced on the strand surface of the scaffold via heating/cooling in tetraglycol without significant deterioration in physical properties. The BMP-2 adsorbed on3D-PLSS-BMPwas continuously released from LSS over a period of 32 d. The LSS can be a microtopographical cue for improved focal cell adhesion, proliferation, and osteogenic differentiation.In vitrocell culture andin vivoanimal studies demonstrated the biological (bioactive BMP-2) and physical (microrough structure) mechanisms of3D-PLSS-BMPfor accelerated bone regeneration. Thus, bioactive molecule-immobilized 3D printing scaffold with LSS represents a promising physically and biologically activated bone graft as well as an advanced tool for widespread application in clinical and research fields.

Nanoclay-Modified Hyaluronic Acid Microspheres for Bone Induction by Sustained rhBMP-2 Delivery.

Microspheres (MSs) are ideal candidates as biological scaffolds loading with growth factors or cells for bone tissue engineering to repair irregular alveolar bone defects by minimally invasive injection. However, the high initial burst release of growth factor and low cell attachment limit the application of microspheres. The modification of microspheres often needs expensive experiments facility or complex chemical reactions, which is difficult to achieve and may bring other problems. In this study, a sol-grade nanoclay, laponite XLS is used to modify the surface of MSs to enhance its affinity to either positively or negatively charged proteins and cells without changing the interior structure of the MSs. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is used as a representation of growth factor to check the osteoinduction ability of laponite XLS-modified MSs. By modification, the protein sustained release, cell loading, and osteoinduction ability of MSs are improved. Modified by 1% laponite XLS, the MSs can not only promote osteogenic differentiation of MC3T3-E1 cells by themselves, but also enhance the effect of the rhBMP-2 below the effective dose. Collectively, the study provides an easy and viable method to modify the biological behavior of microspheres for bone tissue regeneration.

[Comparative study of the effects of intramedullary nail fixation and minimally invasive percutaneous plate internal fixation technique on platelet activation and serum transforming growth factor-ß1(TGF-ß) 1 and bone morphogenetic protein-2 (BMP-2) in patients with tibial and fibular fracture].

OBJECTIVE: To investigate the effect of intramedullary nail fixation (IMN) and minimally invasive percutaneous plate internal fixation (MIPPO) techniques on tibiofibular fractures and their effect on platelet activation and serum transforming growth factor-ß1 (TGF-ß1) and bone morphogenetic protein-2 (BMP-2). METHODS: Total of 105 patients with tibiofibular fractures from February 2019 to February 2020 were selected and divided into 53 cases in the MIPPO group and 52 cases in the IMN group. There were 29 males and 24 females with an average age of (41.74±6.05) years old in MIPPO group;in IMN group, 31 males and 21 females with an average age of (40.59±5.26) years old. The perioperative surgical indexes, postoperative complications, ankle function recovery at 12 months postoperatively, platelet activation indexes at 3 and 7 days preoperatively and postoperatively, and serum TGF-ß1 and BMP-2 levels at 4 and 8 weeks preoperatively and postoperatively were compared between the two groups. RESULTS: The operating time and fracture healing time in the MIPPO group were shorter than those in the IMN group(P<0.05); Compared with the preoperative period, the levels of GMP-140, PAC-1, CD63, and CD61 increased in both groups at 3 and 7 days after surgery, but were lower in the MIPPO group than in the IMN group(P<0.05);the levels of serum TGF-ß1 and BMP-2 increased in both groups at 4 and 8 weeks after surgery compared with the preoperative period, and the postoperative complication rate in the MIPPO group was lower than that in the IMN group(P<0.05);the difference was not statistically significant in the excellent rate of ankle function recovery at 12 months follow-up after surgery between two groups(P>0.05). CONCLUSION: Both intramedullary nail fixation and MIPO technique for treatment of tibia and fibula fractures can improve ankle joint function, but the latter has the advantages of short operation time, fast fracture healing, fewer complications, and light platelet activation. Serum TGF-ß1, BMP-2 level improves quickly.

A Functional Variant Alters the Binding of Bone morphogenetic protein 2 to the Transcription Factor NF-κB to Regulate Bone morphogenetic protein 2 Gene Expression and Chicken Abdominal Fat Deposition.

In this study, we employed a dual-luciferase reporter assay and electrophoretic mobility shift analysis (EMSA) in vitro to explore whether a 12-base pair (bp) insertion/deletion (InDel) variant (namely g.14798187_14798188insTCCCTGCCCCCT) within intron 2 of the chicken BMP2 gene, which was significantly associated with chicken abdominal fat weight and abdominal fat percentage, is a functional marker and its potential regulatory mechanism. The reporter analysis demonstrated that the luciferase activity of the deletion allele was extremely significantly higher than that of the insertion allele (p < 0.01). A bioinformatics analysis revealed that compared to the deletion allele, the insertion allele created a transcription factor binding site of nuclear factor-kappa B (NF-κB), which exhibited an inhibitory effect on fat deposition. A dual-luciferase reporter assay demonstrated that the inhibitory effect of NF-κB on the deletion allele was stronger than that on the insertion allele. EMSA indicated that the binding affinity of NF-κB for the insertion allele was stronger than that for the deletion allele. In conclusion, the 12-bp InDel chicken BMP2 gene variant is a functional variant affecting fat deposition in chickens, which may partially regulate BMP2 gene expression by affecting the binding of transcription factor NF-κB to the BMP2 gene.

Exosomal mRNAs for Angiogenic-Osteogenic Coupled Bone Repair.

Regenerative medicine in tissue engineering often relies on stem cells and specific growth factors at a supraphysiological dose. These approaches are costly and may cause severe side effects. Herein, therapeutic small extracellular vesicles (t-sEVs) endogenously loaded with a cocktail of human vascular endothelial growth factor A (VEGF-A) and human bone morphogenetic protein 2 (BMP-2) mRNAs within a customized injectable PEGylated poly (glycerol sebacate) acrylate (PEGS-A) hydrogel for bone regeneration in rats with challenging femur critical-size defects are introduced. Abundant t-sEVs are produced by a facile cellular nanoelectroporation system based on a commercially available track-etched membrane (TM-nanoEP) to deliver plasmid DNAs to human adipose-derived mesenchymal stem cells (hAdMSCs). Upregulated microRNAs associated with the therapeutic mRNAs are enriched in t-sEVs for enhanced angiogenic-osteogenic regeneration. Localized and controlled release of t-sEVs within the PEGS-A hydrogel leads to the retention of therapeutics in the defect site for highly efficient bone regeneration with minimal low accumulation in other organs.

Changes in Expression in BMP2 and Two Closely Related Genes in Guinea Pig Retinal Pigment Epithelium during Induction and Recovery from Myopia.

PURPOSE: We previously reported differential gene expression of the bone morphogenetic protein 2 (Bmp2) in guinea pig retinal pigment epithelium (RPE) after 1 day of hyperopic defocus, imposed with a negative contact lens (CLs). The study reported here sought to obtain insights into the temporal profiles of gene expression changes in Bmp2, as well as those of two closely related genes, the inhibitor of DNA binding 3 (Id3) and Noggin (Nog), both during myopia induction and when the CL treatment was terminated to allow recovery from induced myopia. METHODS: To induce myopia, 2-week-old pigmented guinea pigs (New Zealand strain, n = 8) wore monocular -10 diopter (D) rigid gas-permeable (RGP) CLs for one week, while the other eye served as a control. Ocular measurements were made at baseline, 3 days, and 7 days after the initiation of CL wear, with treatment then being terminated and additional measurements being made after a further 3 days, 1 week, and 2 weeks. Spherical equivalent refractive errors (SERs), axial length (AL), choroidal thickness (ChT), and scleral thickness (ScT) data were collected using retinoscopy, optical biometry (Lenstar), and spectral domain optical coherence tomography (SD-OCT), respectively. RPE samples were collected from both eyes of the guinea pigs after either 1 day or 1 week of CL wear or 1 day or 2 weeks after its termination, and RNA was subsequently isolated and subjected to quantitative real-time PCR (qRT-PCR) analyses, targeting the Bmp2, Id3, and Nog genes. RESULTS: Mean interocular differences (treated-control) in AL and SER were significantly different from baseline after 3 and 7 days of CL wear, consistent with induced myopia (p < 0.001 for all cases). Termination of CL wear resulted in the normalization (i.e., recovery) of the ALs and SERs of the treated eyes within 7 days, and the earlier significant ChT thinning with CL wear (p = 0004, day 7) was replaced by rapid thickening, which remained significant on day 7 (p = 0.009) but had normalized by day 14. The ChT changes were much smaller in magnitude than the AL changes in both phases. Interocular differences in the ScT showed no significant changes. The Bmp2 and Id3 genes were both significantly downregulated with CL wear, after 1 day (p = 0.012 and 0.016) and 7 days (p = 0.002 and 0.005), while Bmp2 gene expression increased and Nog gene expression decreased after the termination of CL wear, albeit transiently, which was significant on 1 day (p = 0.004 and 0.04) but not 2 weeks later. No change in Id3 gene expression was observed over the latter period. Conclusions: The above patterns of myopia induction and recovery validate this negative RGP-CL model as an alternative to traditional spectacle lens models for guinea pigs. The defocus-driven, sign-dependent changes in the expression of the Bmp2 gene in guinea pig RPE are consistent with observations in chicks and demonstrate the important role of BMP2 in eye growth regulation.

Simvastatin prevents BMP-2 driven cell migration and invasion by suppressing oncogenic DNMT1 expression in breast cancer cells.

Both epigenetic and genetic changes in the cancer genome act simultaneously to promote tumor development and metastasis. Aberrant DNA methylation, a prime epigenetic event, is often observed in various cancer types. The elevated DNA methyltransferase 1 (DNMT1) enzyme creates DNA hypermethylation at CpG islands to drive oncogenic potential. This study emphasized to decipher the molecular mechanism of endogenous regulation of DNMT1 expression for finding upstream signaling molecules. Cancer database analyses found an upregulated DNMT1 expression in most cancer types including breast cancer. Overexpression of DNMT1 showed an increased cell migration, invasion, and stemness potential whereas 5-azacytidine (DNMT1 inhibitor) and siRNA mediated knockdown of DNMT1 exhibited inhibition of such cancer activities in breast cancer MDA-MB-231 and MCF-7 cells. Infact, cancer database analyses further found a positive correlation of DNMT1 transcript with both cholesterol pathway regulatory genes and BMP signaling molecules. Experimental observations documented that the cholesterol-lowering drug, simvastatin decreased DNMT1 transcript as well as protein, whereas BMP-2 treatment increased DNMT1 expression in breast cancer cells. In addition, expression of various key cholesterol regulatory genes was found to be upregulated in response to BMP-2 treatment. Moreover, simvastatin inhibited BMP-2 induced DNMT1 expression in breast cancer cells. Thus, this study for the first time reveals that both BMP-2 signaling and cholesterol pathways could regulate endogenous DNMT1 expression in cancer cells.

MiR-20a promotes osteogenic differentiation in bone marrow-derived mesenchymal stem/stromal cells and bone repair of the maxillary sinus defect model in rabbits.

Introduction: This study aimed to determine whether miR-20 promoted osteogenic differentiation in bone marrow-derived mesenchymal stem/stromal cells (BMSCs) and accelerated bone formation in the maxillary sinus bone defect model in rabbits. Methods: BMSCs were transfected with miR-20a or anti-miR-20a for 12 h, followed by detection of RUNX2, Sp7 mRNA, bone morphogenetic protein 2 (BMP2), and RUNX2 protein expression. Alkaline phosphatase (ALP) activity and Alizarin Red S staining were used to detect calcified nodule deposition. In the rabbit maxillary sinus bone defect model, miR-20a loaded with AAV and BMP2 protein were mixed with Bio-Oss bone powder for filling the bone defect. At 4 weeks and 8 weeks, bone density was detected by cone beam computed tomography (CBCT), and new bone, osteoblasts, and collagen type 1 were evaluated by hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining. Results: Overexpression of miR-20a enhanced the mRNA and protein levels of BMP2, RUNX2, and SP7, the activity of ALP, and the levels of matrix mineralization, whereas the levels and activity of the aforementioned factors were decreased by anti-miR-20a treatment of BMSCs. Furthermore, miR-20a significantly increased the bone density, the number of osteoblasts, and the secretion of collagen type 1 in bone defects compared with Bio-Oss bone powder in the rabbit maxillary sinus bone defect model. Conclusion: Overall, miR-20a can induce osteogenic differentiation in BMSCs and accelerate bone formation of maxillary sinus defects in rabbits.

Tissue engineering at the dentin-pulp interface using human treated dentin scaffolds conditioned with DMP1 or BMP2 plasmid DNA-carrying calcium phosphate nanoparticles.

Hard dental tissue pathologies, such as caries, are conventionally managed through replacement by tooth-colored inert biomaterials. Tissue engineering provides novel treatment approaches to regenerate lost dental tissues based on bioactive materials and/or signaling molecules. While regeneration in the form of reparative dentin (osteo-dentin) is feasible, the recapitulation of the tubular microstructure of ortho-dentin and its special features is sidelined. This study characterized in vitro, and in vivo human EDTA-treated, freeze-dried dentin matrices (HTFD scaffolds) conditioned with calcium phosphate nanoparticles (NPs) bearing plasmids encoding dentinogenesis-inducing factors (pBMP2/NPs or pDMP1/NPs). The uptake and transfection efficiency of the synthesized NPs on dental pulp stem cells (DPSCs) increased in a concentration- and time-dependent manner, as evaluated qualitatively by confocal laser microscopy and transmission electron microscopy, and quantitatively by flow cytometry, while, in parallel, cell viability decreased. HTFD scaffolds conditioned with the optimal transfectability-to-viability concentration at 4 µg Ca/mL of each of the pBMP2/NPs or pDMP1/NPs preserved high levels of cell viability, evidenced by live/dead staining in vitro and caused no adverse reactions after implantation on C57BL6 mice in vivo. HTFD/NPs constructs induced rapid and pronounced odontogenic shift of the DPSCs, as evidenced by relevant gene expression patterns of RunX2, ALP, BGLAP, BMP-2, DMP-1, DSPP by real-time PCR, and acquirement of polarized meta-mitotic phenotype with cellular protrusions entering the dentinal tubules as visualized by scanning electron microscopy. Taken together, HTFD/NPs constitute a promising tool for customized reconstruction of the ortho-dentin/odontoblastic layer barrier and preservation of pulp vitality. STATEMENT OF SIGNIFICANCE: In clinical dentistry, the most common therapeutic approach for the reconstruction of hard dental tissue defects is the replacement by resin-based restorative materials. Even modern bioactive materials focus on reparative dentinogenesis, leading to amorphous dentin-bridge formation in proximity to the pulp. Therefore, the natural microarchitecture of tubular ortho-dentin is not recapitulated, and the sensory and defensive role of odontoblasts is sidelined. This study approaches the reconstruction at the dentin-pulp interface using a construct of human treated dentin (HTFD) scaffold and plasmid-carrying nanoparticles (NPs) encoding dentinogenic factors (DMP-1 or BMP-2) with excellent in vitro and in vivo properties. As a future perspective, the HTFD/NPs constructs could act as bio-fillings for personalized reconstruction of the dentin-pulp interface.

Icariin Has a Synergistic Effect on the Osteoinductivity of Bone Morphogenetic Protein 2 at Ectopic Sites.

OBJECTIVE: Establishing biocompatible, biodegradable, osteoconductive, and osteoinductive bone materials remains a challenging subject in the research of bone healing and bone regeneration. Previously, we demonstrated the osteogenic and osteoconductive effects of biomimetic calcium phosphate (BioCaP) incorporating with Icariin and/or bone morphogenetic protein 2 (BMP-2) at orthotopic sites. METHODS: By implanting the BioCaP granules incorporated Icariin and/or BMP-2 into the dorsal subcutaneous pockets of adult male Sprague-Dawley (S-D) rats (6-7 weeks old), we investigated the osteoinductive efficacy of the samples. Micro-computed tomography(micro-CT) observations and histological slices were used to verify the osteoinduction of this system on the 2nd and 5th week. Statistical significances was evaluated using Turkey's post hoc test of one-way analysis of variance. RESULTS: The osteoinduction of the BioCaP incorporated with BMP-2 or both agents was confirmed as expected. BioCaP with Icariin alone could not generate bone formation at an ectopic sites. Nevertheless, co-administration of Icariin increased bone mineral density (BMD; p < 0.01) (628mg HA/cm3 vs 570mg HA/cm3 ) and completely changed the distribution of newly formed bone when compared with the granules with BMP-2 alone, even though there was no significant difference in the volume of newly formed bone. In contrast, the BioCaP with both agents (37.86%) had significantly fewer remaining materials than the other groups by the end of the fifth week (53.22%, 53.62% and 48.22%) (p < 0.01). CONCLUSION: The co-administration of Icariin and BMP-2 increased BMD changed the distribution of newly formed bone, and reduced the amount of remaining materials. Therefore, Icariin can stimulate BMP-2 when incorporated into BioCaP granules at ectopic sites, which makes it useful for bone tissue engineering.

Amniotic membrane attenuates heterotopic ossification following high-dose bone morphogenetic protein-2 treatment of segmental bone defects.

Treatment of large bone defects with supraphysiological doses of bone morphogenetic protein-2 (BMP-2) has been associated with complications including heterotopic ossification (HO), inflammation, and pain, presumably due to poor spatiotemporal control of BMP-2. We have previously recapitulated extensive HO in our rat femoral segmental defect model by treatment with high-dose BMP-2 (30 µg). Using this model and BMP-2 dose, our objective was to evaluate the utility of a clinically available human amniotic membrane (AM) around the defect space for guided bone regeneration and reduction of HO. We hypothesized that AM surrounding collagen sponge would attenuate heterotopic ossification compared with collagen sponge alone. In vitro, AM retained more BMP-2 than a synthetic poly(ε-caprolactone) membrane through 21 days. In vivo, as hypothesized, the collagen + AM resulted in significantly less heterotopic ossification and correspondingly, lower total bone volume (BV), compared with collagen sponge alone. Although bone formation within the defect was delayed with AM around the defect, by 12 weeks, defect BVs were equivalent. Torsional stiffness was significantly reduced with AM but was equivalent to that of intact bone. Collagen + AM resulted in the formation of dense fibrous tissue and mineralized tissue, while the collagen group contained primarily mineralized tissue surrounded by marrow-like structures. Especially in conjunction with high doses of growth factor delivered via collagen sponge, these findings suggest AM may be effective as an overlay adjacent to bone healing sites to spatially direct bone regeneration and minimize heterotopic ossification.

Dopamine/DOPAC-assisted immobilization of bone morphogenetic protein-2 loaded Heparin/PEI nanogels onto three-dimentional printed calcium phosphate ceramics for enhanced osteoinductivity and osteogenicity.

Nowadays, the three-dimensional (3D) printed calcium phosphate (CaP) ceramics have well-designed geometric structure, but suffer from relative weak osteoinductivity. Surface modification by incorporating bone morphogenetic protein-2 (BMP2) onto scaffolds is considered as an efficient approach to improve their bioactivity. However, high dose and uncontrolled burst release of BMP2 may cause undesired side effect. In the present study, porous BCP ceramics with inverse face-centred cube structure prepared by digital light processing (DLP)-based 3D printing technique were used as the substrates. BMP2 proteins were loaded in the self-assembled Heparin/PEI nanogels (NP/BMP2), and then immobilized onto BCP substrates through the intermediate mussel-derived bioactive dopamine and dihydroxyphenylacetic acid (DA/DOPAC) coating layers to construct functional BCP/layer/NP/BMP2 scaffolds. Our results showed that Heparin/PEI nanogel was a potent delivery system for BMP2, and BCP/layer/NP/BMP2 scaffolds exhibited the high loading capacity, controlled release rate, and sustained local delivery of BMP2. In vitro cell experiments with bone marrow stromal cells (BMSCs) found that BCP/layer/NP/BMP2 could promote cell proliferation, facilitate cell spreading, accelerate cell migration, up-regulate expression of osteogenic genes, and improve synthesis of osteoblast-related proteins. Moreover, the murine intramuscular implantation model suggested that BCP/layer/NP/BMP2 had a superior osteoinductive capacity, and the rat femoral condyle defect repair model showed that BCP/layer/NP/BMP2 could enhance in situ bone repair and regeneration. These findings demonstrate that the incorporation of BMP2 loaded Heparin/PEI nanogels to 3D printed scaffolds holds great promise in fabricating bone graft with a superior biological performance for orthopedic application.

ß-Ecdysterone Enhanced Bone Regeneration Through the BMP-2/SMAD/RUNX2/Osterix Signaling Pathway.

Bone defects are a global public health problem. However, the available methods for inducing bone regeneration are limited. The application of traditional Chinese herbs for bone regeneration has gained popularity in recent years. ß-ecdysterone is a plant sterol similar to estrogen, that promotes protein synthesis in cells; however, its function in bone regeneration remains unclear. In this study, we investigated the function of ß-ecdysterone on osteoblast differentiation and bone regeneration in vitro and in vivo. MC3T3-E1 cells were used to test the function of ß-ecdysterone on osteoblast differentiation and bone regeneration in vitro. The results of the Cell Counting Kit-8 assay suggested that the proliferation of MC3T3-E1 cells was promoted by ß-ecdysterone. Furthermore, ß-ecdysterone influenced the expression of osteogenesis-related genes, and the bone regeneration capacity of MC3T3-E1 cells was detected by polymerase chain reaction, the alkaline phosphatase (ALP) test, and the alizarin red test. ß-ecdysterone could upregulate the expression of osteoblastic-related genes, and promoted ALP activity and the formation of calcium nodules. We also determined that ß-ecdysterone increased the mRNA and protein levels of components of the BMP-2/Smad/Runx2/Osterix pathway. DNA sequencing further confirmed these target effects. ß-ecdysterone promoted bone formation by enhancing gene expression of the BMP-2/Smad/Runx2/Osterix signaling pathway and by enrichment biological processes. For in vivo experiments, a femoral condyle defect model was constructed by drilling a bone defect measuring 3 mm in diameter and 4 mm in depth in the femoral condyle of 8-week-old Sprague Dawley male rats. This model was used to further assess the bone regenerative functions of ß-ecdysterone. The results of micro-computed tomography showed that ß-ecdysterone could accelerate bone regeneration, exhibiting higher bone volume, bone surface, and bone mineral density at each observation time point. Immunohistochemistry confirmed that the ß-ecdysterone also increased the expression of collagen, osteocalcin, and bone morphogenetic protein-2 in the experiment group at 4 and 8 weeks. In conclusion, ß-ecdysterone is a new bone regeneration regulator that can stimulate MC3T3-E1 cell proliferation and induce bone regeneration through the BMP-2/Smad/Runx2/Osterix pathway. This newly discovered function of ß-ecdysterone has revealed a new direction of osteogenic differentiation and has provided novel therapeutic strategies for treating bone defects.

Spatially and Temporally Controllable BMP-2 and TGF-ß3 Double Release From Polycaprolactone Fiber Scaffolds via Chitosan-Based Polyelectrolyte Coatings.

Temporally and spatially controlled growth factor release from a polycaprolactone fiber mat, which also provides a matrix for directional cell colonization and infiltration, could be a promising regenerative approach for degenerated tendon-bone junctions. For this purpose, polycaprolactone fiber mats were coated with tailored chitosan-based nanogels to bind and release the growth factors bone morphogenetic protein 2 (BMP-2) and transforming growth factor-ß3 (TGF-ß3), respectively. In this work we provide meaningful in vitro data for the understanding of the drug delivery performance and sterilizability of novel implant prototypes in order to lay the foundation for in vivo testing. ELISA-based in vitro release studies were used to investigate the spatial and temporal control of release, as well as the influence of radiation sterilization on protein activity and release behavior. Layer-by-layer coatings based on BMP-2-containing chitosan tripolyphosphate nanogel particles and negatively charged alginate showed a good sustainment of BMP-2 release from chemically modified polycaprolactone fiber mats. Release control improved with increasing layer numbers. The approach of controlling the release via a barrier of cross-linked chitosan azide proved less promising. By using a simple, partial immersion-based dip-coating process, it was possible to apply opposing gradients of the growth factors BMP-2 and TGF-ß3. Final radiation sterilization of the growth factor-loaded implant prototypes resulted in a radiation dose-correlated degradation of the growth factors, which could be prevented by lyophilization into protective matrices. For the manufacture of sterile implants, the growth factor loading step must probably be carried out under aseptic conditions. The layer-by-layer coated implant prototypes provided sustained release from opposing gradients of the growth factors BMP-2 and TGF-ß3 and thus represent a promising approach for the restoration of tendon-bone defects.

Preparation of BMP-2/PDA-BCP Bioceramic Scaffold by DLP 3D Printing and its Ability for Inducing Continuous Bone Formation.

Digital light processing (DLP)-based 3D printing is suitable to fabricate bone scaffolds with small size and high precision. However, the published literature mainly deals with the fabrication procedure and parameters of DLP printed bioceramic scaffold, but lacks the subsequent systematic biological evaluations for bone regeneration application. In this work, a biphasic calcium phosphate (BCP) macroporous scaffold was constructed by DLP-based 3D printing technique. Furthermore, bone morphogenetic protein-2 (BMP-2) was facilely incorporated into this scaffold through a facile polydopamine (PDA) modification process. The resultant scaffold presents an interconnected porous structure with pore size of ∼570 µm, compressive strength (∼3.6 MPa), and the self-assembly Ca-P/PDA nanocoating exhibited excellent sustained-release property for BMP-2. Notably, this BMP-2/PDA-BCP scaffold presents favorable effects on the adhesion, proliferation, osteogenic differentiation, and mineralization of bone marrow stromal cells (BMSCs). Furthermore, in vivo experiments conducted on rats demonstrated that the scaffolds could induce cell layer aggregation adjacent to the scaffolds and continuous new bone generation within the scaffold. Collectively, this work demonstrated that the BMP-2/PDA-BCP scaffold is of immense potential to treat small craniofacial bone defects in demand of high accuracy.

Site-Directed Immobilization of an Engineered Bone Morphogenetic Protein 2 (BMP2) Variant to Collagen-Based Microspheres Induces Bone Formation In Vivo.

For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2's bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.

Local administration of low doses of exogenous BMP2 and leptin promotes ectopic bone regeneration in leptin-deficient mice.

BACKGROUND: Obesity and leptin deficiency are associated with compromised bone regeneration. OBJECTIVE: This study aims to investigate the role of locally administrated low-dose BMP2+leptin on bone regeneration in leptin-deficient obese (ob/ob) mice. METHODS: Wildtype (WT) and ob/ob mice were divided into 3 groups (4 mice/group): BMP2 (5 µg) group, BMP2+low-dose leptin (1 µg) group, and BMP2+high-dose leptin (2.5 µg) group. WT mice were used as control mice. An equal size absorbable collagen sponge was prepared by loading the BMP2 or/and leptin and implanted subcutaneously. After 19 days, samples were collected and analyzed by micro-CT and H&E staining. RESULTS: No significant difference in bone regeneration among the three groups in WT mice. Quantification of newly formed bone parameters from micro-CT and H&E staining showed that low-dose BMP2 treatment formed less new bone in ob/ob mice compared to WT. BMP2+low-dose leptin treatment substantially rescued the compromised bone regeneration in ob/ob mice up to the level in WT mice. However, the BMP2 and high dose of leptin failed to rescue the compromised bone regeneration in ob/ob mice. CONCLUSION: Our findings suggest that a combination of the low-dose BMP2 and leptin could be a strategy to promote osteogenesis in obese populations with leptin deficiency.

NF-κB activation impedes the transdifferentiation of hypertrophic chondrocytes at the growth plate of mouse embryos in diabetic pregnancy.

BACKGROUND: Diabetes mellitus could cause numerous complications and health problems including abnormality of endochondral bone formation during embryogenesis. However, the underlying mechanisms still remain obscure. METHODS: Streptozotoci (STZ) was injected to induce pregestational diabetes mellitus (PGDM) mouse model. The femurs of E18.5 mouse embryos from control and PGDM groups were harvested. Morphological staining was implemented to determine the abnormality of the bone development. The expressions of the key genes participating in osteogenesis (e.g., Sox9, Runx2, and Osterix), the NF-κB signaling molecules (e.g., P50, P65, IκBα), and the corresponding regulatory factors (e.g., Bmp2, phospho-p38) were evaluated by immunofluorescence, quantitative PCR and western blot. Finally, in vitro chondrocyte differentiation model was employed to verify the role of NF-κB on the expressions of chondro-osteogenic markers. RESULTS: Alcian blue/alizarin red double staining and H&E staining demonstrated the restriction of skeletal development and relatively extended hypertrophic zone at growth plate in E18.5 STZ-induced diabetic mouse embryos compared to the control. Immunofluorescent staining and qPCR showed that Sox9 expression increased, while Runx2 and Osterix expressions decreased in the growth plate of the offspring of PGDM mice. Immunofluorescence of P65 manifested the activation of NF-κB signaling in growth plate in PGDM mouse embryos. Furthermore, the relatively extended hypertrophic zone was also observed in the growth plate of the NF-κB-activated transgenic mice, as well as the activated p65 up-regulated the expression of Bmp2 and p-p38. In ATDC5 cells, we could observe the high glucose up-regulated the P50 and P65 expressions and down-regulated IκBα expression, but the high glucose-activated NF-κB signaling could be reversed by addition of Bay (inhibitor of NF-κB signaling). The expression changes of Bmp2, Sox9 and Runx2 in presence of high glucose were resumed too. CONCLUSION: Our data revealed that NF-κB signaling was involved in mediation effects of dysfunctional trans-differentiation of hypertrophic chondrocytes in the embryonic growth plate induced by maternal diabetic mellitus.

Enhanced bone regeneration via spatiotemporal and controlled delivery of a genetically engineered BMP-2 in a composite Hydrogel.

Scaffolds functionalized with bone morphogenetic protein-2 (BMP-2) have shown great potential for bone regeneration. However, structural instability and the necessity for supra-physiological dose have thus far limited practical applications for BMP-2. Protein modification and site-specific covalent immobilization of BMP-2 to carrier materials might be optimal strategies to overcome these problems. Here, we report a broadly applicable strategy where the polyhistidine tag-T4 Lysozyme (His6-T4L) was genetically fused at the N-terminus of BMP-2 and used as a protein spacer, which on one hand enhanced protein solubility and stability, and on the other hand mediated site-specific covalent anchoring of BMP-2 upon binding to nickel-chelated nitrilotriacetic acid (Ni-NTA) microparticles (denoted as MPs-His6-T4L-BMP2) to further maximize its rescued activity. We also constructed a novel gelatin-based hydrogel that was crosslinked by transglutaminase (TG) and tannic acid (TA). This hydrogel, when incorporated with MPs-His6-T4L-BMP2, displayed excellent in-situ injectability, thermosensitivity, adhesiveness and improved mechanical properties. The effective loading mode led to a controlled and long-term sustained release of His6-T4L-BMP2, thereby resulting in enhancement of bone regeneration in a critical-sized bone defect. We believe that the protein modification strategy proposed here opens up new route not only for BMP-2 applications, but can be used to inform novel uses for other macromolecules.

Augmentation of implant surfaces with BMP-2 in a revision setting : effects of local and systemic bisphosphonate.

AIMS: We wanted to evaluate the effects of a bone anabolic agent (bone morphogenetic protein 2 (BMP-2)) on an anti-catabolic background (systemic or local zoledronate) on fixation of allografted revision implants. METHODS: An established allografted revision protocol was implemented bilaterally into the stifle joints of 24 canines. At revision surgery, each animal received one BMP-2 (5 µg) functionalized implant, and one raw implant. One group (12 animals) received bone graft impregnated with zoledronate (0.005 mg/ml) before impaction. The other group (12 animals) received untreated bone graft and systemic zoledronate (0.1 mg/kg) ten and 20 days after revision surgery. Animals were observed for an additional four weeks before euthanasia. RESULTS: No difference was detected on mechanical implant fixation (load to failure, stiffness, energy) between local or systemic zoledronate. Addition of BMP-2 had no effect on implant fixation. In the histomorphometric evaluation, implants with local zoledronate had more area of new bone on the implant surface (53%, p = 0.025) and higher volume of allograft (65%, p = 0.007), whereas implants in animals with systemic zoledronate had the highest volume of new bone (34%, p = 0.003). Systemic zoledronate with BMP-2 decreased volume of allograft by 47% (p = 0.017). CONCLUSION: Local and systemic zoledronate treatment protects bone at different stages of maturity; local zoledronate protects the allograft from resorption and systemic zoledronate protects newly formed bone from resorption. BMP-2 in the dose evaluated with experimental revision implants was not beneficial, since it significantly increased allograft resorption without a significant compensating anabolic effect. Cite this article: Bone Joint Res 2021;10(8):488-497.