Recombinant Bone Morphogenetic Protein-2 Research Articles

The Role Of BMPs in the Regulation of Osteoclasts Resorption and Bone Remodeling: From Experimental Models to Clinical Applications.

In response to mechanical forces and the aging process, bone in the adult skeleton is continuously remodeled by a process in which old and damaged bone is removed by bone-resorbing osteoclasts and subsequently is replaced by new bone by bone-forming cells, osteoblasts. During this essential process of bone remodeling, osteoclastic resorption is tightly coupled to osteoblastic bone formation. Bone-resorbing cells, multinuclear giant osteoclasts, derive from the monocyte/macrophage hematopoietic lineage and their differentiation is driven by distinct signaling molecules and transcription factors. Critical factors for this process are Macrophage Colony Stimulating Factor (M-CSF) and Receptor Activator Nuclear Factor-κB Ligand (RANKL). Besides their resorption activity, osteoclasts secrete coupling factors which promote recruitment of osteoblast precursors to the bone surface, regulating thus the whole process of bone remodeling. Bone morphogenetic proteins (BMPs), a family of multi-functional growth factors involved in numerous molecular and signaling pathways, have significant role in osteoblast-osteoclast communication and significantly impact bone remodeling. It is well known that BMPs help to maintain healthy bone by stimulating osteoblast mineralization, differentiation and survival. Recently, increasing evidence indicates that BMPs not only help in the anabolic part of bone remodeling process but also significantly influence bone catabolism. The deletion of the BMP receptor type 1A (BMPRIA) in osteoclasts increased osteoblastic bone formation, suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone-formation activity during bone remodeling. The dual effect of BMPs on bone mineralization and resorption highlights the essential role of BMP signaling in bone homeostasis and they also appear to be involved in pathological processes in inflammatory disorders affecting bones and joints. Certain BMPs (BMP2 and -7) were approved for clinical use; however, increased bone resorption rather than formation were observed in clinical applications, suggesting the role BMPs have in osteoclast activation and subsequent osteolysis. Here, we summarize the current knowledge of BMP signaling in osteoclasts, its role in osteoclast resorption, bone remodeling, and osteoblast–osteoclast coupling. Furthermore, discussion of clinical application of recombinant BMP therapy is based on recent preclinical and clinical studies.

Assessment of BMP responses in an in vitro model of acute ethanol toxicity

The assay presented here was designed to assess the immediate effects of ethanol (EtOH) exposure on intracellular signaling activated by BMPs (Bone Morphogenetic Proteins). Previous reports of the relationship between EtOH exposure and BMP-dependent signaling have primarily assessed the expression of individual BMPs, changes in BMP target genes or effects on the phosphorylation level of key downstream mediators after days or weeks of in vivo EtOH exposure. What happens to BMP-stimulated signaling immediately following exposure to EtOH remains largely unexplored. Here, the early events of BMP-evoked intracellular signaling were examined in an in vitro model of acute EtOH toxicity. The BMP/Ethanol Stimulation Assay involved first stimulating cultured cells with recombinant BMPs. BMP-evoked intracellular signaling was then allowed to develop for 30 minutes. Next, the cells were exposed to a range of EtOH concentrations for an additional 30 minutes. Finally, the cultures were processed for Western blot analysis or immunofluorescent labeling.This short-term assay:• Permits investigation of EtOH exposure during the initial signaling events downstream of BMP receptor activation• Enables assessment of how the presence of BMPs might protect against cellular injury caused by toxic EtOH levels

HYBRID IMPLANT BASED ON NATURAL MATERIALS WITH SILICATE CERAMIC PARTICLES AS A CARRIER OF RECOMBINANT BMP-2: HISTOLOGICAL STUDY OF REPARATIVE OSTEOGENESIS DURING IMPLANTATION INTO CRITICAL-SIZED CRANIAL DEFECTS IN MICE

For the first time it was shown that calcium-magnesium silicate ceramic diopside can be an effective carrier of recombinant bone morphogenetic protein 2, causing strong repara-tive osteogenesis in the area of critical size skull defects in mice when inserted as a hy-brid implant that also includes demineralized bone matrix as a scaffold.

A Pre-clinical Standard Operating Procedure for Evaluating Orthobiologics in an In Vivo Rat Spinal Fusion Model.

The rat animal model is a cost effective and reliable model used in spinal pre-clinical research. Complications from various surgical procedures in humans often arise that were based on these pre-clinical animal models. Therefore safe and efficacious pre-clinical animal models are needed to establish continuity into clinical trials. A Standard Operating Procedure (SOP) is a validated method that allows researchers to safely and carefully replicate previously successful surgical techniques. Thus, the aim of this study is to describe in detail the procedures involved in a common rat bilateral posterolateral intertransverse spinal fusion SOP used to test the efficacy and safety different orthobiologics using a collagen-soaked sponge as an orthobiologic carrier. Only two orthobiologics are currently FDA approved for spinal fusion surgery which include recombinant bone morphogenetic protein 2 (rhBMP-2), and I-FACTOR. While there are many additional orthobiologics currently being tested, one way to show their safety profile and gain FDA approval, is to use well established pre-clinical animal models. A preoperative, intraoperative, and postoperative surgical setup including specific anesthesia and euthanasia protocols are outlined. Furthermore, we describe different postoperative methods used to validate the spinal fusion SOP, which include μCT analysis, histopathology, biomechanical testing, and blood analysis. This SOP can help increase validity, transparency, efficacy, and reproducibly in future rat spinal fusion surgery procedures.

Prefabricated 3D-Printed Tissue-Engineered Bone for Mandibular Reconstruction: A Preclinical Translational Study in Primate.

The advent of three dimensionally (3D) printed customized bone grafts using different biomaterials has enabled repairs of complex bone defects in various in vivo models. However, studies related to their clinical translations are truly limited. Herein, 3D printed poly(lactic-co-glycolic acid)/β-tricalcium phosphate (PLGA/TCP) and TCP scaffolds with or without recombinant bone morphogenetic protein −2 (rhBMP-2) coating were utilized to repair primate’s large-volume mandibular defects and compared efficacy of prefabricated tissue-engineered bone (PTEB) over direct implantation (without prefabrication). 18F-FDG PET/CT was explored for real-time monitoring of bone regeneration and vascularization. After 3-month’s prefabrication, the original 3D-architecture of the PLGA/TCP-BMP scaffold was found to be completely lost, while it was properly maintained in TCP-BMP scaffolds. Besides, there was a remarkable decrease in the PLGA/TCP-BMP scaffold density and increase in TCP-BMP scaffolds density during ectopic (within latissimus dorsi muscle) and orthotopic (within mandibular defect) implantation, indicating regular bone formation with TCP-BMP scaffolds. Notably, PTEB based on TCP-BMP scaffold was successfully fabricated with pronounced effects on bone regeneration and vascularization based on radiographic, 18F-FDG PET/CT, and histological evaluation, suggesting a promising approach toward clinical translation.

Incorporation of hydroxyapatite into collagen scaffolds enhances the therapeutic efficacy of rhBMP-2 in a weight-bearing femoral defect model

Traditional methods to promote bone healing, such as autologous bone grafting, and therapeutic strategies involving recombinant bone morphogenetic proteins (BMPs), such as BMP-2 and BMP-7, have been associated with performance limitations and dose-related safety concerns, respectively. Thus, there is an unmet need for osteoinductive bone graft substitutes that can enhance the therapeutic efficacy of rhBMP-2 and facilitate dose-reduction approaches. This study aimed to determine the therapeutic efficacy of rhBMP-2 delivered using a collagen-only scaffold (CO) (representative of clinical standards), a collagen-hydroxyapatite scaffold (CHA), and a thermoresponsive hyaluronic acid hydrogel doped with gelatin and laminin (HyA-GL). Efficacy over 14 weeks was evaluated using 2 mm segmental femoral defects in skeletally mature female F344 rats, internally fixated using a 1.25 mm-thick polyetheretherketone plate. Empty defects failed to bridge but administration of rhBMP-2 using either CO or CHA scaffolds led to successful bridging, while using HyA-GL resulted in non-union. CHA scaffolds induced enhanced and stiffer new bone formation compared to both CO and HyA-GL scaffolds. Limited luminal remodeling occurred in both CO and CHA groups. Taken together, this study has determined that a collagen-hydroxyapatite scaffold can enhance the therapeutic efficacy of rhBMP-2 compared to collagen-only scaffolds routinely used in standard clinical practice.

Osteogenic Competence and Potency of the Bone Induction Principle: Inductive Substrates That Initiate "Bone: Formation by Autoinduction".

The de novo induction of bone has always been a fascinating phenomenon, keeping skeletal reconstructionists and cellular developmental biologists continuously engaged to finally provide a molecular and cellular approach to the induction of bone formation. A significant advancement was made by the purification and cloning of the human recombinant bone morphogenetic proteins, members of the transforming growth factor-β supergene family. Human bone morphogenetic proteins are powerful inducers of bone in animal models including nonhuman primates. Translation in clinical contexts has however, proven to be surprisingly difficult. This review also describes the significant induction of bone formation by the human transforming growth factor-β3 when implanted in heterotopic intramuscular sites of the Chacma baboon Papio ursinus. Large mandibular defects implanted with 250 mg human transforming growth factor-β3 in human patients showed significant osteoinduction; however, the induction of bone was comparatively less than the induction of bone in P ursinus once again highlighting the conundrum of human osteoinduction: is the bone induction principle failing clinical translation?

Effect of Bone Morphogenetic Protein-2 in the Treatment of Long Bone Non-Unions.

Background: Delayed fracture healing continues to cause significant patient morbidity and an economic burden to society. Biological stimulation of non-unions includes application of recombinant bone morphogenetic protein-2 (rhBMP-2). However, rhBMP-2 use continues to be a matter of controversy as literature shows scarce evidence for treatment effectiveness. Questions: The objective of this study was to evaluate the effectiveness of rhBMP-2 treatment on long bone non-unions measuring union rate and time to union. Furthermore, we assess risk factors for treatment failure. Methods and patients: A total of 91 patients with non-unions of long bones were treated with rhBMP-2 (n = 72) or standard care without BMP (n = 19) at our institution. Patient characteristics, comorbidities, nicotine consumption, and complications were recorded. Bone healing was assessed by plane X-rays and clinical examination. Patients were followed up with for 24 months. Results: Overall, there was significantly faster bone healing after rhBMP-2 application compared to the no-BMP group (p < 0.001; HR = 2.78; 95% CI 1.4–5.6). Union rates differed significantly between rhBMP-2 compared to the no-BMP group (89% vs. 47%; p < 0.001). At the humerus, there was neither a significantly higher union rate in the rhBMP-2 (83%) compared to the no-BMP group (50%) (p = 0.26; n = 12) nor a faster bone healing with a median time of 9 months in both groups (HR = 2.01; 95% CI 0.49–8.61; p = 0.315). The 33 femora treated using rhBMP-2 healed significantly faster than 9 femora in the no-BMP group (HR = 2.93; 95% CI 1.00–8.4; p = 0.023) with significant differences in union rate with 85% and 44%, respectively (p = 0.022). Regarding tibia non-unions, 25 out of 27 (93%) healed with a median of 9 months after rhBMP-2 application with no significant difference in the no-BMP group (33%) in time to union (p = 0.097) but a significantly higher union rate (p = 0.039). There was no effect of comorbidities, age, sex, soft tissue damage, or nicotine use on time to union, union rate, or secondary interventions. Conclusion: Consistent with the literature, overall, significantly higher union rates with reduced time to union were achieved after rhBMP-2 application. Femoral and tibial non-unions in particular seem to profit from rhBMP-2 application.

Therapy of delayed consolidation of mandible fractures

The aim of the study is to improve the effectiveness of treatment for delayed consolidation of mandibular fractures by developing and clinical testing of integrated therapy using growth factors and bone morphogenetic protein-2. Clinical material is based on the study of 62 patients with the problem of mandibular fractures consolidation. The examinations of bone density were carried out on a cone-beamed computerized tomograph Planmeca Promax 3D (Finland) with an X-ray emitter of 85 kV 5-7 mA on the day 7 and 21 of the study. The evaluation of the density of jaw bone tissue was conducted by U. Lekholm and G. Zarb (1985) classifications, and by S. Mish (1990). Bone density of more than 850 units was considered as an intact dense bone, the values from 350 to 850 units - as a relatively intact, loose bone, and less than 350 Hounsfield units - local osteoporosis. Statistical processing of materials was carried out using standard methods of variation statistics using the STATISTIKA (ver. 6.1; Statsoft Inc., USA, No. AGAR 909E415822FA). Use in the therapy of delayed consolidation of mandible fractures of the developed complex, which includes strontium ranelate – a drug with osteotropic action, antioxidant Mexidol, immunomodulator of recombinant human interleukin-2, and a local introduction of fibrin-enriched platelets containing numerous growth factors along the fissure of mandible fracture, or recombinant bone morphogenetic protein-2 in more complicated cases. The improvement of reparative processes was in 58 patients. In any case there was no need to carry out additional surgical interventions for osteosynthesis or bone «grafting». The basic program on care of patients with a delayed consolidation of mandible fractures should affect all pathogenesis components of this complication: it should include osteotropic drugs, antioxidants and immunomodulators. Involvement of platelet-rich fibrin in the integrated therapy positively affects the quality of bone regenerate and treatment terms, ensuring the normalization of clinical and laboratory parameters up to the day 21. The best optimization of reparative osteogenesis in the complex therapy of delayed consolidation is facilitated by the additional injection of recombinant human bone morphogenetic protein-2. This tactic allowed to ensure its favorable course by the type of initially delayed adhesion in all patients (8 out of 8).

Bone Morphogenetic Protein-2 Rapidly Heals Two Distinct Critical Sized Segmental Diaphyseal Bone Defects in a Porcine Model.

Segmental bone defects (SBDs) are devastating injuries sustained by warfighters and are difficult to heal. Preclinical models that accurately simulate human conditions are necessary to investigate therapies to treat SBDs. We have developed two novel porcine SBD models that take advantage of similarities in bone healing and immunologic response to injury between pigs and humans. The purpose of this study was to investigate the efficacy of Bone Morphogenetic Protein-2 (BMP-2) to heal a critical sized defect (CSD) in two novel porcine SBD models. Two CSDs were performed in Yucatan Minipigs including a 25.0-mm SBD treated with intramedullary nailing (IMN) and a 40.0-mm SBD treated with dual plating (ORIF). In control animals, the defect was filled with a custom spacer and a bovine collagen sponge impregnated with saline (IMN25 Cont, n = 8; ORIF40 Cont, n = 4). In experimental animals, the SBD was filled with a custom spacer and a bovine collage sponge impregnated with human recombinant BMP-2 (IMN25 BMP, n = 8; ORIF40 BMP, n = 4). Healing was quantified using monthly modified Radiographic Union Score for Tibia Fractures (mRUST) scores, postmortem CT scanning, and torsion testing. BMP-2 restored bone healing in all eight IMN25 BMP specimens and three of four ORIF40 BMP specimens. None of the IMN25 Cont or ORIF40 Cont specimens healed. mRUST scores at the time of sacrifice increased from 9.2 (±2.4) in IMN25 Cont to 15.1 (±1.0) in IMN25 BMP specimens (P < .0001). mRUST scores increased from 8.2 (±1.1) in ORIF40 Cont to 14.3 (±1.0) in ORIF40 BMP specimens (P < .01). CT scans confirmed all BMP-2 specimens had healed and none of the control specimens had healed in both IMN and ORIF groups. BMP-2 restored 114% and 93% of intact torsional stiffness in IMN25 BMP and ORIF40 BMP specimens. We have developed two porcine CSD models, including fixation with IMN and with dual-plate fixation. Porcine models are particularly relevant for SBD research as the porcine immunologic response to injury closely mimics the human response. BMP-2 restored healing in both CSD models, and the effects were evident within the first month after injury. These findings support the use of both porcine CSD models to investigate new therapies to heal SBDs.

Bioactive chitosan biguanidine-based injectable hydrogels as a novel BMP-2 and VEGF carrier for osteogenesis of dental pulp stem cells

Nowadays, vascularization and mineralization of bone defects is the main bottleneck in the bone regeneration field that is needed to be overcome and developed. Here, we prepared novel in-situ formed injectable hydrogels based on chitosan biguanidine and carboxymethylcellulose loaded with vascular endothelial growth factor (VEGF) and recombinant Bone morphogenetic protein 2 (BMP-2) and studied its influence on osteoblastic differentiation of dental pulp stem cells (DPSCs). The sequential release behavior of the VEGF and BMP-2 from hydrogels adjusted with the pattern of normal human bone growth. MTT assay exhibited that these hydrogels were non-toxic and significantly increased DPSCs proliferation. The Real-time PCR and Western blot analysis on CG11/BMP2-VEGF showed significantly higher gene and protein expression of ALP, COL1α1, and OCN. These results were confirmed by mineralization assay by Alizarin Red staining and Alkaline phosphatase enzyme activity. Based on these evaluations, these hydrogel holds potential as an injectable bone tissue engineering platform.

Platelet-Rich Fibrin Increases BMP2 Expression in Oral Fibroblasts via Activation of TGF-β Signaling.

Solid platelet-rich fibrin (PRF), consisting of coagulated plasma from fractionated blood, has been proposed to be a suitable carrier for recombinant bone morphogenetic protein 2 (BMP2) to target mesenchymal cells during bone regeneration. However, whether solid PRF can increase the expression of BMPs in mesenchymal cells remains unknown. Proteomics analysis confirmed the presence of TGF-β1 but not BMP2 in PRF lysates. According to the existing knowledge of recombinant TGF-β1, we hypothesized that PRF can increase BMP2 expression in mesenchymal cells. To test this hypothesis, we blocked TGF-β receptor 1 kinase with SB431542 in gingival fibroblasts exposed to PRF lysates. RT-PCR and immunoassays confirmed that solid PRF lysates caused a robust SB431542-dependent increase in BMP2 expression in gingival fibroblasts. Additionally, fractions of liquid PRF, namely platelet-poor plasma (PPP) and the buffy coat (BC) layer, but not heat-denatured PPP (Alb-gel), greatly induced the expression of BMP2 in gingival fibroblasts. Even though PRF has no detectable BMPs, PRF lysates similar to recombinant TGF-β1 had the capacity to provoke canonical BMP signaling, as indicated by the nuclear translocation of Smad1/5 and the increase in its phosphorylation. Taken together, our data suggest that PRF can activate TGF-β receptor 1 kinase and consequently induce the production of BMP2 in cells of the mesenchymal lineage.

Positive Effects of Three-Dimensional Collagen-Based Matrices on the Behavior of Osteoprogenitors.

Recent research has demonstrated that reinforced three-dimensional (3D) collagen matrices can provide a stable scaffold for restoring the lost volume of a deficient alveolar bone. In the present study, we aimed to comparatively investigate the migratory, adhesive, proliferative, and differentiation potential of mesenchymal stromal ST2 and pre-osteoblastic MC3T3-E1 cells in response to four 3D collagen-based matrices. Dried acellular dermal matrix (DADM), hydrated acellular dermal matrix (HADM), non-crosslinked collagen matrix (NCM), and crosslinked collagen matrix (CCM) did all enhance the motility of the osteoprogenitor cells. Compared to DADM and NCM, HADM and CCM triggered stronger migratory response. While cells grown on DADM and NCM demonstrated proliferative rates comparable to control cells grown in the absence of a biomaterial, cells grown on HADM and CCM proliferated significantly faster. The pro-proliferative effects of the two matrices were supported by upregulated expression of genes regulating cell division. Increased expression of genes encoding the adhesive molecules fibronectin, vinculin, CD44 antigen, and the intracellular adhesive molecule-1 was detected in cells grown on each of the scaffolds, suggesting excellent adhesive properties of the investigated biomaterials. In contrast to genes encoding the bone matrix proteins collagen type I (Col1a1) and osteopontin (Spp1) induced by all matrices, the expression of the osteogenic differentiation markers Runx2, Alpl, Dlx5, Ibsp, Bglap2, and Phex was significantly increased in cells grown on HADM and CCM only. Short/clinically relevant pre-coating of the 3D biomaterials with enamel matrix derivative (EMD) or recombinant bone morphogenetic protein-2 (rBMP-2) significantly boosted the osteogenic differentiation of both osteoprogenitor lines on all matrices, including DADM and NCM, indicating that EMD and BMP-2 retained their biological activity after being released from the matrices. Whereas EMD triggered the expression of all osteogenesis-related genes, rBMP-2 upregulated early, intermediate, and late osteogenic differentiation markers except for Col1a1 and Spp1. Altogether, our results support favorable influence of HADM and CCM on the recruitment, growth, and osteogenic differentiation of the osteoprogenitor cell types. Furthermore, our data strongly support the biofunctionalization of the collagen-based matrices with EMD or rBMP-2 as a potential treatment modality for bone defects in the clinical practice.

BMP6 Mediates BMP2-Increased Human Trophoblast Invasion

TGF-β superfamily proteins play divergent roles in regulating human extravillous trophoblast (EVT) invasion and their coordinated effects are essential for adequate placentation during pregnancy 1. Bone morphogenetic protein 2 (BMP2), which belongs to the BMP subfamily of TGF-β superfamily, has been shown to promote human EVT invasion and the acquisition of endothelial-like phenotype 2,3. It has been reported that BMP2 promotes EVT invasion by up-regulating Activin A, a growth factor which also belongs to TGF-β superfamily. However, whether BMP6 mediates the pro-invasive effect of BMP2 has yet to be determined. Herein, we firstly treated immortalized trophoblast cells (HTR8/SVneo) with recombinant BMP2 protein for 6 and 24 hrs, and our bulk-RNA sequencing results demonstrated significantly increased BMP6 mRNA levels after BMP2 treatment. Furthermore, we confirmed the up-regulatory effects of BMP2 on BMP6 mRNA and protein levels in both HTR8/SVneo and primary EVTs isolated from first-trimester villi. Notably, siRNA-mediated down-regulation of BMP6 significantly attenuated both basal and BMP2-induced cell invasion in HTR8/SVneo cells as measured by Matrigel-coated transwell invasion assay. In summary, our results firstly demonstrated the up-regulatory effect of BMP2 on BMP6 expression in human trophoblasts and identified the mediation role of BMP6 in BMP2-promoted EVT invasion, suggesting the interplay between BMP subfamily members during EVT invasion regulation. Our ongoing research focusing the underlying molecular mechanisms and signaling pathways could further benefit the advancement of diagnostic and therapeutic strategies for EVT invasion dysregulation-related pregnancy disorders, e.g., pre-eclampsia. Reference: (1) Li Yan et al., Trends Endocrinol Metab 2021 18: S1043-2760(20)30266-6. (2) Hong-Jin Zhao et al., FASEB J 2020;34(2):3151-3164. (3) Hong-Jin Zhao et al., Cell Death Dis 2018;9(2):174.

Osteoinductivity and biomechanical assessment of a 3D printed demineralized bone matrix-ceramic composite in a rat spine fusion model

We recently developed a recombinant growth factor-free bone regenerative scaffold composed of stoichiometric hydroxyapatite (HA) ceramic particles and human demineralized bone matrix (DBM) particles (HA-DBM). Here, we performed the first pre-clinical comparative evaluation of HA-DBM relative to the industry standard and established positive control, recombinant human bone morphogenetic protein-2 (rhBMP-2), using a rat posterolateral spinal fusion model (PLF). Female Sprague–Dawley rats underwent bilateral L4-L5 PLF with implantation of the HA-DBM scaffold or rhBMP-2. Fusion was evaluated using radiography and blinded manual palpation, while biomechanical testing quantified the segmental flexion-extension range-of-motion (ROM) and stiffness of the fused segments at 8-weeks postoperatively. For mechanistic studies, pro-osteogenic gene and protein expression at 2-days and 1-, 2-, and 8-weeks postoperatively was assessed with another cohort. Unilateral fusion rates did not differ between the HA-DBM (93%) and rhBMP-2 (100%) groups; however, fusion scores were higher with rhBMP-2 (p = 0.008). Both treatments resulted in significantly reduced segmental ROM (p < 0.001) and greater stiffness (p = 0.009) when compared with non-operated controls; however, the degree of stabilization was significantly higher with rhBMP-2 treatment relative to the HA-DBM scaffold. In the mechanistic studies, PLGA and HA scaffolds were used as negative controls. Both rhBMP-2 and HA-DBM treatments resulted in significant elevations of several osteogenesis-associated genes, including Runx2, Osx, and Alp. The rhBMP-2 treatment led to significantly greater early, mid, and late osteogenic markers, which may be the mechanism in which early clinical complications are seen. The HA-DBM scaffold also induced osteogenic gene expression, but primarily at the 2-week postoperative timepoint. Overall, our findings show promise for this 3D-printed composite as a recombinant growth factor-free bone graft substitute for spinal fusion. Statement of significanceDespite current developments in bone graft technology, there remains a significant void in adequate materials for bone regeneration in clinical applications. Two of the most efficacious bone graft options are the gold-standard iliac crest bone graft and recombinant human-derived bone morphogenetic protein-2 (rhBMP-2), available commercially as Infuse™. Although efficacious, autologous graft is associated with donor-site morbidity, and Infuse™ has known side effects related to its substantial host inflammatory response, possibly associated with a immediate, robust osteoinductive response. Hence, there is a need for a bone graft substitute that provides adequate osteogenesis without associated adverse events. This study represents a significant step in the design of off-the-shelf growth factor-free devices for spine fusion.

Osseointegration evaluation of UHMWPE and PEEK-based scaffolds with BMP-2 using model of critical-size cranial defect in mice and push-out test

It the present study the push-out mechanical test was adopted for mouse model of implantation in critical-size cranial defects to evaluate the effectiveness of implant-skull fusion. As implants, disks of porous ultra-high molecular weight polyethylene (UHMWPE) and polyetheretherketone (PEEK) with hydroxylapatite (HA) with and without loading of recombinant bone morphogenetic protein-2 (BMP-2) were used. Implantation results were evaluated using histology and micro-computed tomography (micro-CT). In the case of both UHMWPE/HA and PEEK/HA, BMP-2 loading resulted in a significant increase in the amount of bone tissue in the implantation area, especially at the edges of the defect, and an increase in the value of BV/TV (bone volume/tissue volume) during micro-CT. There was a high correlation of BV/TV values with the maximum load and elastic modulus measured during the puch-out test. The maximum load values showed good convergence within the groups and are comparable to the maximum load values obtained by other authors in the rat model of implantation in critical-size cranial defects. An adapted push-out test can be used to evaluate the quality of osseointegration of the implanted materials.

Copper-Free Azide–Alkyne Cycloaddition for Peptide Modification of Alginate Hydrogels

Alginate, a biocompatible polymer naturally derived from algae, is widely used as a synthetic analogue of the extracellular matrix in tissue engineering. Integrin-binding peptide motifs, including RGD, a derivative of fibronectin, are typically grafted to the alginate polymer through carbodiimide reactions between peptide amines and alginate uronic acids. However, lack of chemo-selectivity of carbodiimide reactions can lead to side reactions that lower peptide bioactivity. To overcome these limitations, we developed an approach for copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC)-mediated conjugation of azide-modified adhesive peptides (azido-cyclo-RGD, Az-cRGD) onto alginate. Successful conjugation of azide-reactive cyclooctynes onto alginates using a heterobifunctional crosslinker was confirmed by azido-coumarin fluorescent assay, NMR, and through click reactions with azide-modified fluorescent probes. Compared to cyclo-RGD peptides directly conjugated to alginate polymers with standard carbodiimide chemistry, Az-cyclo-RGD peptides exhibited higher bioactivity, as demonstrated by cell adhesion and proliferation assays. Finally, Az-cRGD peptides enhanced the effects of recombinant bone morphogenetic proteins on inducing osteogenesis of osteoblasts and bone marrow stromal stem cells in 3D alginate gels. SPAAC-mediated click approaches for peptide-alginate bioconjugation overcome the limitations of previous alginate bioconjugation approaches and potentially expand the range of ligands that can be grafted to alginate polymers for tissue engineering applications.

Internal Radioulnar Fixation for Treatment of Nonunion of Proximal Radius and Ulna Fractures in a Toy Breed Dog

AbstractIn this case report, we describe an alternative surgical procedure to treat proximal radius and ulnar nonunion in a toy breed dog. A 14-month-old, Maltese cross-breed dog was referred after previous treatment with external and internal fixation had failed, resulting in a nonunion of a fracture of the proximal radius and ulna with the proximal radius fragment too small and friable to be used for fixation. A craniomedial approach was made to debride the radius nonunion site and a second approach to the lateral aspect of the ulna was made. The fracture was realigned and a titanium locking plate was applied in bridging fashion, fixed to the proximal ulnar fragment with three locking screws in the most proximal plate holes, a fourth screw was inserted in the mid-shaft of the distal ulnar fragment and three locking screws were inserted in the distal most holes of the plate through the distal ulna to engage the distal radial fragment. A recombinant bone morphogenetic protein 2 graft was inserted into the radius and ulna fracture sites. The dog had a successful clinical and radiographic outcome with bridging of the defect 4 weeks postoperatively and complete callus formation 8 weeks postoperatively. Implants have undergone dynamization and then removal. Use of a locking plate as an internal fixator achieving fixation of the proximal ulna and distal radius can be considered an option for the treatment of proximal radioulnar nonunions with a small proximal radial fragment.

Optogenetic Control of the BMP Signaling Pathway.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGFβ) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic, and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise activation of the BMP signaling pathway. Here, we report the development of an optogenetic BMP signaling system (optoBMP) that enables rapid induction of the canonical BMP signaling pathway driven by illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signaling pathway through variable light stimulation. Optogenetic control of BMP signaling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.

The Bone Morphogenetic Protein Pathway: The Osteoclastic Perspective.

Bone health crucially relies on constant bone remodeling and bone regeneration, both tightly controlled processes requiring bone formation and bone resorption. Plenty of evidence identifies bone morphogenetic proteins (BMP) as major players in osteoblast differentiation and thus, bone formation. However, in recent past years, researchers also increasingly reported on the pivotal role of these multi-functional growth factors in osteoclast formation and activity. This review aims to summarize the current knowledge of BMP signaling within the osteoclast lineage, its role in bone resorption, and osteoblast–osteoclast coupling. Furthermore, subsequent clinical implications for recombinant BMP therapy will be discussed in view of recent preclinical and clinical studies.