Μg Of Bone Morphogenetic Protein-2 Research Articles

Bisphosphonates do not affect healing of a critical-size defect in estrogen-deficient mice

Bisphosphonates (BP) are anti-resorptive drugs that are widely used to prevent bone loss in osteoporosis. Since inhibition of bone resorption will cause a decrease in bone formation through a process called coupling, it is hypothesized that extended treatment protocols may impair bone healing. In this study, β-tri‑calcium-phosphate (βTCP) ceramics were inserted into critical-size long bone defects in estrogen-deficient mice under BP therapy. The study assessed the benefits of coating the ceramics with Bone Morphogenetic Protein-2 (BMP2) and an engineered BMP2 analogue (L51P) that inactivates BMP antagonists on the healing process, implant resorption, and bone formation.Female NMRI mice (11–12 weeks of age) were ovariectomized (OVX) or sham operated. Eight weeks later, after the manifestation of ovariectomy-induced osteoporotic bone changes, BP therapy with Alendronate (ALN) was commenced. After another five weeks, a femoral critical-size defect was generated, rigidly fixed, and βTCP-cylinders loaded with 0.25 μg or 2.5 μg BMP2, 2.5 μg L51P, and 0.25 μg BMP2/2.5 μg L51P, respectively, were inserted. Unloaded βTCP-cylinders were used as controls. Femora were collected six and twelve weeks post-implantation.Histological and micro-computer tomography (MicroCT) evaluation revealed that insertion of cylinders coated with 2.5 μg BMP2 accelerated fracture repair and induced significant bone formation compared to controls (unloaded cylinders or coated with 2.5 μg L51P, 0.25 μg BMP2) already six weeks post-implantation, independent of estrogen-deficiency and BP therapy. The simultaneous administration of BMP2 and L51P (0.25 μg BMP2/2.5 μg L51P) did not promote fracture healing six and twelve weeks post-implantation. Moreover, new bone formation within the critical-size defect was directly linked to the removal of the βTCP-implant in all experimental groups. No evidence was found that long-term therapy with ALN impaired the resorption of the implanted graft. However, osteoclast transcriptome signature was elevated in sham and OVX animals upon treatment with BP, with transcript levels being higher at six weeks than at twelve weeks post-surgery. Furthermore, the transcriptome profile of the developing repair tissue confirmed an accelerated repair process in animals treated with 2.5 μg BMP2 implants. L51P did not increase the bioefficacy of BMP2 in the applied defect model.The present study provides evidence that continuous administration of BP does not inhibit implant resorption and does not alter the kinetics of the healing process of critical-size long bone defects. Furthermore, the BMP2 variant L51P did not enhance the bioefficacy of BMP2 when applied simultaneously to the femoral critical-size defect in sham and OVX mice.

Use of biosilica to improve loading and delivery of bone morphogenic protein 2

The clinical utility of bone morphogenetic protein 2 (BMP2) is limited because of the poor attraction between BMP2 and carriers, resulting in low loading efficiency and initial burst release. Here, the high binding affinity of BMP2 to the biosilica surface was utilized to overcome this limitation. Atomic force microscopy revealed that BMP2 bound nearly 8- and 2-fold more strongly to biosilica-coated hydroxyapatite than to uncoated and plain silica-coated hydroxyapatite, respectively. To achieve controlled release, collagen was introduced between the silica layers on hydroxyapatite, which was optimized by adjusting the collagen concentration and number of layers. The optimal biosilica/collagen formulation induced sustained BMP2 release without compromising loading efficiency. BMP2 combined with the mentioned formulation led to an increase in osteogenesis, as compared to the combination of BMP2 with either biosilica-coated or non-coated hydroxyapatite in vitro. In rat calvarial defect models, the biosilica/collagen-coated hydroxyapatite with 1 μg BMP2 showed 26 % more bone regeneration than the same dose of BMP2-loaded hydroxyapatite and 10.6 % more than hydroxyapatite with 2.5-fold dose of BMP2. Using BMP2 affinity carriers coated with biosilica/collagen allows for more efficacious in situ loading and delivery of BMP2, making them suitable for the clinical application of growth factors through a soaking method.

Effect of Bone Morphogenetic Protein-2 (BMP-2)/Hydroxyapatite/In Situ-Formed Hyaluronan Hydrogel Composites on Bone Formation in a Murine Model of Posterolateral Lumbar Fusion

Several types of calcium phosphate (CaP) biomaterial carriers have been designed to deliver bone morphogenetic protein-2 (BMP-2) to augment spinal fusion in spinal surgery. Here, we evaluated an in situ-formed hydrogel (IFH) constructed from hyaluronan (IFH-HA) combined with a BMP2/hydroxyapatite (HAP) composite in bone formation in a murine model of posterolateral lumbar fusion (PLF). HAP was submerged in HA-tyramine (TA) polymer solution containing horseradish peroxidase (HRP) and 2 µg BMP-2 (BMP2/HA-TA/HRP solution). H2O2 was added to initiate the curing reaction (BMP-2/IFH-HA). phosphate-buffered saline (PBS) was added to the BMP2/HA-TA/HRP solution (BMP-2/HA-TA) instead of H2O2 to evaluate the effectiveness of the curing reaction. HAP immersed in PBS was used as a control. PLF model mice were randomly assigned to receive one these composites (n = 10 each).X-ray images were taken to assess the bone fusion, and microcomputed tomography analysis was conducted to examine new bone formation at the graft site four weeks following surgery. No evidence of fusion was observed four weeks after surgery in the Control or BMP2/HA-TA group. In contrast, the BMP2/IFH-HA group exhibited newly formed bone between the transverse processes and bone union in coronal sections. Relative to the Control and BMP2/HA-TA groups, the BMP2/IFH-HA group showed significantly greater bone volume. The BMP2/IFH-HA group also showed significantly elevated bone mineral content relative to the BMP2/HA-TA group. A composite comprising BMP2/HAP and IFH-HA, thus, enhanced the new bone formation in a murine model of PLF, suggesting its promise for augmenting spinal fusion.

Bone marrow mesenchymal stem cells with low dose bone morphogenetic protein 2 enhances scaffold-based spinal fusion in a porcine model.

High doses bone morphogenetic protein 2 (BMP-2) have resulted in a series of complications in spinal fusion. We previously established a polyelectrolyte complex (PEC) carrier system that reduces the therapeutic dose of BMP-2 in both rodent and porcine spinal fusion models. This study aimed to evaluate the safety and efficacy of the combination of bone marrow mesenchymal stem cells (BMSCs) and low dose BMP-2 delivered by PEC for bone regeneration in a porcine model of anterior lumbar interbody spinal fusion (ALIF) application. Six Yorkshire pigs underwent a tri-segmental (L2/L3; L3/L4; L4/L5) ALIF in four groups, namely: (a) BMSCs+25μg BMP-2/PEC (n=9), (b) 25μg BMP-2/PEC (n=3), (c) BMSCs (n=3), and (d) 50μg BMP-2/absorbable collagen sponge (n=3). Fusion outcomes were evaluated by radiography, biomechanical testing, and histological analysis after 12weeks. Mean radiographic scores at 12weeks were 2.7, 2.0, 1.0, and 1.0 for Groups 1 to 4, respectively. μ-CT scanning, biomechanical evaluation, and histological analysis demonstrated solid fusion and successful bone regeneration in Group 1. In contrast, Group 2 showed inferior quality and slow rate of fusion, and Groups 3 and 4 failed to fuse any of the interbody spaces. There was no obvious evidence of seroma formation, implant rejection, or any other complications in all groups. The results suggest that the combination of BMSCs and low dose BMP-2/PEC could further lower down the effective dose of the BMP-2 and be used as a bone graft substitute in the large animal ALIF model.

Personalized Baghdadite scaffolds: stereolithography, mechanics and in vivo testing

An ongoing challenge in the field of orthopedics is to produce a clinically relevant synthetic ceramic scaffold for the treatment of ‘critical-sized’ bone defects, which cannot heal without intervention. We had developed a bioactive ceramic (baghdadite, Ca₃ZrSi₂O₉) and demonstrated its outstanding bioactivity using traditional manufacturing techniques. Here, we report on the development of a versatile stereolithography printing technology that enabled fabrication of anatomically-shaped and -sized Baghdadite scaffolds. We assessed the in vivo bioactivity of these scaffolds in co-delivering of bone morphogenetic protein-2 (BMP2) and zoledronic acid (ZA) through bioresorbable coatings to induce bone formation and increase retention in a rat model of heterotopic ossification. Micro-computed tomography, histology, mechanical tests pre- and post-implantation, and mechanical modelling were used to assess bone ingrowth and its effects on the mechanics of the scaffolds. Bone ingrowth and the consequent mechanical properties of the scaffolds improved with increasing BMP2 dose. Co-delivery of ZA with BMP2 further improved this outcome. The significant bone formation within the scaffolds functionalized with 10 µg BMP2 and 2 µg ZA made them 2.3 × stiffer and 2.7 × stronger post-implantation and turned these inherently brittle scaffolds into a tough and deformable material. The effects of bone ingrowth on the mechanical properties of scaffolds were captured in a mechanical model that can be used in future clinical studies for non-destructive evaluation of scaffold's stiffness and strength as new bone forms. These results support the practical utilization of our versatile stereolithographic printing methods and BMP2/ZA functionalization to create fit-for-purpose personalized implants for clinical trials. Statement of significanceIn this study, we addressed a long-standing challenge of developing a ceramic printing technology that enables fabrication of customizable anatomically-shaped and -sized bioceramic scaffolds with precise internal architectures using an inexpensive desktop printer. We also addressed another challenge related to delivery of pharmaceuticals. BMP2, currently available as a bone-inducing bioactive protein, is clinically administered in a collagen scaffold that has limited moldability and poor mechanical properties. The comparably stiffer and stronger 3D printed personalized Baghdadite scaffolds developed here can be readily functionalized with bioresorbable coatings containing BMP2 ± ZA. These innovations considerably improve on the prior art and are scalable for use in human surgery.

Human salivary histatin-1 (Hst1) promotes bone morphogenetic protein 2 (BMP2)-induced osteogenesis and angiogenesis.

Large‐volume bone defects can result from congenital malformation, trauma, infection, inflammation and cancer. At present, it remains challenging to treat these bone defects with clinically available interventions. Allografts, xenografts and most synthetic materials have no intrinsic osteoinductivity, and so an alternative approach is to functionalize the biomaterial with osteoinductive agents, such as bone morphogenetic protein 2 (BMP2). Because it has been previously demonstrated that human salivary histatin‐1 (Hst1) promotes endothelial cell adhesion, migration and angiogenesis, we examine here whether Hst1 can promote BMP2‐induced bone regeneration. Rats were given subcutaneous implants of absorbable collagen sponge membranes seeded with 0, 50, 200 or 500 μg Hst1 per sample and 0 or 2 μg BMP2 per sample. At 18 days postsurgery, rats were sacrificed, and implanted regional tissue was removed for micro computed tomography (microCT) analyses of new bone (bone volume, trabecular number and trabecular separation). Four samples per group were decalcified and subjected to immunohistochemical staining to analyze osteogenic and angiogenic markers. We observed that Hst1 increased BMP2‐induced new bone formation in a dose‐dependent manner. Co‐administration of 500 μg Hst1 and BMP2 resulted in the highest observed bone volume and trabecular number, the lowest trabecular separation and the highest expression of osteogenic markers and angiogenic markers. Our results suggest that coadministration of Hst1 may enhance BMP2‐induced osteogenesis and angiogenesis, and thus may have potential for development into a treatment for large‐volume bone defects.

Induction of periosteal bone formation by intraosseous BMP-2 injection in a mouse model of osteogenesis imperfecta.

PurposeSurgical interventions are routinely performed on children with osteogenesis imperfecta (OI) to stabilize long bones, often post fracture. We speculated that a combination of intramedullary reaming and intraosseous injection of recombinant bone morphogenetic protein-2 (BMP-2) could enhance periosteal ossification and ultimately cortical thickness and strength. This approach was conceptually tested in a preclinical model of genetic bone fragility.MethodsSix experimental groups were tested including no treatment, intramedullary reaming, and reaming with 5 µg BMP-2 injection performed in the tibiae of both wild type (WT) and Col1a2G610C/+ (OI, Amish mutation) mice. Bone formation was examined at a two-week time point in ex vivo specimens by micro-computed tomography (microCT) analysis and histomorphometry with a dynamic bone label.ResultsMicroCT data illustrated increases in tibial cortical thickness with intramedullary reaming alone (Saline) and reaming plus BMP-2 injection (BMP-2) compared to no intervention controls. In the OI mice, the periosteal bone increase was not statistically significant with Saline but there was an increase of +192% (p = 0.053) with BMP-2 injection. Dynamic histomorphometry on calcein label was used to quantify new woven bone formation; while BMP-2 induced greater bone formation than Saline, the anabolic response was blunted overall in the OI groups.ConclusionsThese data indicate that targeting the intramedullary compartment via reaming and intraosseous BMP-2 delivery can lead to gains in cortical bone parameters. It is suggested that the next step is to validate safety and functional improvements in a clinical OI setting.

Injectable BMP-2 delivery system based on collagen-derived microspheres and alginate induced bone formation in a time- and dose-dependent manner.

The aim of the current study was to reduce the clinically used supra-physiological dose of bone morphogenetic protein-2 (BMP-2) (usually 1.5 mg/mL), which carries the risk of adverse events, by using a more effective release system. A slow release system, based on an injectable hydrogel composed of BMP-2-loaded recombinant collagen-based microspheres and alginate, was previously developed. Time- and dose-dependent subcutaneous ectopic bone formation within this system and bone regeneration capacity in a calvarial defect model were investigated. BMP-2 doses of 10 µg, 3 µg and 1 µg per implant (50 µg/mL, 15 µg/mL and 5 µg/mL, respectively) successfully induced ectopic bone formation subcutaneously in rats in a time- and dose-dependent manner, as shown by micro-computed tomography (µCT) and histology. In addition, the spatio-temporal control of BMP-2 retention was shown for 4 weeks in vivo by imaging of fluorescently-labelled BMP-2. In the subcritical calvarial defect model, µCT revealed a higher bone volume for the 2 µg of BMP-2 per implant condition (50 µg/mL) as compared to the lower dose used (0.2 µg per implant, 5 µg/mL). Complete defect bridging was obtained with 50 µg/mL BMP-2 after 8 weeks. The BMP-2 concentration of 5 µg/mL was not sufficient to heal a calvarial defect faster than the empty defect or biomaterial control without BMP-2. Overall, this injectable BMP-2 delivery system showed promising results with 50 µg/mL BMP-2 in both the ectopic and calvarial rat defect models, underling the potential of this composite hydrogel for bone regeneration therapies.

CSA-90 Promotes Bone Formation and Mitigates Methicillin-resistant Staphylococcus aureus Infection in a Rat Open Fracture Model.

Infection of open fractures remains a significant cause of morbidity and mortality to patients worldwide. Early administration of prophylactic antibiotics is known to improve outcomes; however, increasing concern regarding antimicrobial resistance makes finding new compounds for use in such cases a pressing area for further research. CSA-90, a synthetic peptidomimetic compound, has previously demonstrated promising antimicrobial action against Staphylococcus aureus in rat open fractures. However, its efficacy against antibiotic-resistant microorganisms, its potential as a therapeutic agent in addition to its prophylactic effects, and its proosteogenic properties all require further investigation. (1) Does prophylactic treatment with CSA-90 reduce infection rates in a rat open fracture model inoculated with S aureus, methicillin-resistant S aureus (MRSA), and methicillin-resistant Staphylococcus epidermidis (MRSE) as measured by survival, radiographic union, and deep tissue swab cultures? (2) Does CSA-90 reduce infection rates when administered later in the management of an open fracture as measured by survival, radiographic union, and deep tissue swab cultures? (3) Does CSA-90 demonstrate a synergistic proosteogenic effect with bone morphogenetic protein 2 (BMP-2) in a noninfected rat ectopic bone formation assay as assessed by micro-CT bone volume measurement? (4) Can CSA-90 elute and retain its antimicrobial efficacy in vitro when delivered using clinically relevant agents measured using a Kirby-Bauer disc diffusion assay? All in vivo studies were approved by the local animal ethics committee. In the open fracture studies, 12-week-old male Wistar rats underwent open midshaft femoral fractures stabilized with a 1.1-mm Kirschner wire and 10 µg BMP-2 ± 500 µg CSA-90 was applied to the fracture site using a collagen sponge along with 1 x 10 colony-forming units of bacteria (S aureus/MRSA/MRSE; n = 10 per group). In the delayed treatment study, débridement and treatment with 500 µg CSA-90 were performed at Day 1 and Day 5 after injury and bacterial insult (S aureus). All animals were reviewed daily for signs of local infection and/or sepsis. An independent, blinded veterinarian reviewed twice-weekly radiographs, and rats showing osteolysis and/or declining overall health were culled at his instruction. The primary outcome of both fracture studies was fracture infection, incorporating survival, radiographic union, and deep tissue swab cultures. For the ectopic bone formation assay, 0 to 10 µg BMP-2 and 0 to 500 µg CSA-90 were delivered on a collagen sponge into bilateral quadriceps muscle pouches of 8-week-old rats (n = 10 per group). Micro-CT quantification of bone volume and descriptive histologic analysis were performed for all in vivo studies. Modified Kirby-Bauer disc diffusion assays were used to quantify antimicrobial activity in vitro using four different delivery methods, including bone cement. Infection was observed in none of the MRSA inoculated open fractures treated with CSA-90 with 10 of 10 deep tissue swab cultures negative at the time of cull. Median survival was 43 days (range, 11-43 days) in the treated group versus 11 days (range, 8-11 days) in the untreated MRSA inoculated group (p < 0.001). However, delayed débridement and treatment of open fractures with CSA-90 at either Day 1 or Day 5 did not prevent infection, resulting in early culls by Day 21 with positive swab cultures (10 of 10 for each time point). Maximal ectopic bone formation was achieved with 500 μg CSA-90 and 10 μg BMP-2 (mean volume, 9.58 mm; SD, 7.83), creating larger bone nodules than formed with 250 μg CSA-90 and 10 μg BMP-2 (mean volume, 1.7 mm; SD, 1.07; p < 0.001). Disc diffusion assays showed that CSA-90 could successfully elute from four potential delivery agents including calcium sulphate (mean zone of inhibition, 11.35 mm; SD, 0.957) and bone cement (mean, 4.67 mm; SD, 0.516). CSA-90 shows antimicrobial action against antibiotic-resistant Staphylococcal strains in vitro and in an in vivo model of open fracture infection. The antimicrobial properties of CSA-90 combined with further evidence of its proosteogenic potential make it a promising compound to develop further for orthopaedic applications.

Influence of Mussel-Derived Bioactive BMP-2-Decorated PLA on MSC Behavior in Vitro and Verification with Osteogenicity at Ectopic Sites in Vivo.

Osteoinductive activity of the implant in bone healing and regeneration is still a challenging research topic. Therapeutic application of recombinant human bone morphogenetic protein-2 (BMP-2) is a promising approach to enhance osteogenesis. However, high dose and uncontrolled burst release of BMP-2 may introduce edema, bone overgrowth, cystlike bone formation, and inflammation. In this study, low-dose BMP-2 of 1 μg was used to design PLA-PD-BMP for functionalization of polylactic acid (PLA) implants via mussel-inspired polydopamine (PD) assist. For the first time, the binding property and efficiency of the PD coating with BMP-2 were directly demonstrated and analyzed using an antigen-antibody reaction. The obtained PLA-PD-BMP surface immobilized with this low BMP-2 dose can endow the implants with abilities of introducing strong stem cell adhesion and enhanced osteogenicity. Furthermore, in vivo osteoinduction of the PLA-PD-BMP-2 scaffolds was confirmed by a rat ectopic bone model, which is marked as the "gold standard" for the evidence of osteoinductive activity. The microcomputed tomography, Young's modulus, and histology analyses were also employed to demonstrate that PLA-PD-BMP grafted with 1 μg of BMP-2 can induce bone formation. Therefore, the method in this study can be used as a model system to immobilize other growth factors onto various different types of polymer substrates. The highly biomimetic mussel-derived strategy can therefore improve the clinical outcome of polymer-based medical implants in a facile, safe, and effective way.

Unidirectional BMP2-loaded collagen scaffolds induce chondrogenic differentiation

Microfracture surgery may be improved by the implantation of unidirectional collagen scaffolds that provide a template for mesenchymal stem cells to regenerate cartilage. Incorporation of growth factors in unidirectional scaffolds may further enhance cartilage regeneration. In scaffolds, immobilization of growth factors is required to prolong in vivo activity, to limit diffusion and to reduce the amount of growth factor needed for safe clinical application. We investigated the immobilization of bone morphogenetic protein 2 (BMP2) to unidirectional collagen scaffolds and the effect on in vitro chondrogenesis. C3H10T1/2 cells were seeded on unidirectional collagen scaffolds with and without covalently attached heparin, and with and without incubation with BMP2 (1 and 10 μg), or with BMP2 present in the culture medium (10–200 ng ml−1). Culturing was for 2 weeks and readout parameters included histology, immunohistochemistry, biochemical analysis and molecular biological analysis. The unidirectional pores facilitated the distribution of C3H10T1/2 cells and matrix formation throughout scaffolds. The effective dose of medium supplementation with BMP2 was 100 ng ml−1 (total exposure 1 μg BMP2), and similar production of cartilage-specific molecules chondroitin sulfate (CS) and type II collagen was found for scaffolds pre-incubated with 10 μg BMP2. Pre-incubation with 1 μg BMP2 resulted in less cartilage matrix formation. The conjugation of heparin to the scaffolds resulted in more CS and less type II collagen deposition compared to scaffolds without heparin. In conclusion, unidirectional collagen scaffolds pre-incubated with 10 μg BMP2 supported chondrogenesis in vitro and may be suitable for prolonged cartilage matrix synthesis in vivo.

Titanium implant functionalization with phosphate-containing polymers may favour in vivo osseointegration.

Osseointegration of titanium implants is predictable, but can be improved via surface functionalization. One hundred and twenty implants were installed in parietal bone of 12 domestic pigs and left to heal for 1 or 3months. Five groups were defined according surface treatments: immersion in water (H2 O), 10% polyphosphoric acid (PPA10), 1% phosphorylated pullulan (PPL1), 10% phosphorylated pullulan (PPL10) or 10% phosphorylated pullulan+1μg bone morphogenetic protein-2 (PPL10 BMP). As primary outcome, implant osseointegration was evaluated by quantitative histology, namely peri-implant bone formation (B/T in %) and bone-to-implant contact (BIC in %) for each healing period. The Wilcoxon signed-rank test and Mann-Whitney U-test with α=0.05 were performed. PPL10 and PPA10 groups showed significantly higher B/T and BIC results than the control (H2 O) group at 1-month (p<.05). No significant difference was found between PPL1 and H2 O or between PPL10 BMP and H2 O, irrespective of healing time (1 or 3months) or investigated parameter (B/T and BIC; p>.05). After 3months, no experimental group showed a significant difference compared to the control group (H2 O) for both investigated parameters (B/T and BIC; p>.05). Functionalizing titanium implants with inorganic or organic phosphate-containing polymers at 10wt% concentration may stimulate peri-implant bone formation and implant osseointegration at early healing times.

Evaluation of the effectiveness of a bMSC and BMP-2 polymeric trilayer system in cartilage repair

In this study a poly(lactide-co-glycolide) acid (PLGA) tri-layer scaffold is proposed for cartilage repair. The trilayer system consists of a base layer formed by a tablet of PLGA microspheres, a second layer composed of a microsphere suspension placed on top of the tablet, and the third layer, which constitutes an external electrospun PLGA thin polymeric membrane. Combinations of bone morphogenetic protein-2 (BMP-2) encapsulated in the microspheres of the suspension layer, and bone marrow mesenchymal stem cells (bMSC) seeded on the electrospun membrane, are evaluated by histologic analyses and immunohistochemistry in a critical size osteochondral defect in rabbits. Five experimental groups, including a control group (empty defect), a blank group (blank scaffold), a bMSC treated group, two groups treated with 2.5 μg or 8.5 μg of BMP-2 and another two groups implanted with bMSC-BMP-2 combination are evaluated. The repair area increases throughout the experimental time (24 weeks). The repair observed in the treated groups is statistically higher than in control and blank groups. However, the bMSC-BMP-2 combination does not enhance the BMP-2 response. In conclusion, BMP-2 and bMSC repaired effectively the osteochondral defect in the rabbits. The bMSC-BMP-2 combination did not produce synergism.

Heparin-Based Polyelectrolyte Complex Enhances the Therapeutic Efficacy of Bone Morphogenetic Protein-2 for Posterolateral Fusion in a Large Animal Model.

The study was based on porcine posterolateral fusion model. The study aims to prove that polyelectrolyte complex (PEC) carrier could enhance the efficacy and safety profile of bone morphogenetic protein-2 (BMP-2). BMP-2 was introduced to enhance posterolateral fusion; however, extremely high doses of this molecule were often used which contributed to various complications. This was attributed to the poor modulation capacity of the traditional carrier absorbable collagen sponge (ACS). To reduce the efficacious dose of BMP-2 and its associated complications, heparin-based PEC was introduced. L3/L4 and L5/L6 two-level posterolateral spinal fusion was performed on six pigs using two doses of BMP-2 with PEC or ACS: (1) PEC with 800 μg BMP-2 (n = 2); (2) PEC with 400 μg BMP-2 (n = 2); (3) ACS with 800 μg BMP-2 (n = 1); (4) ACS with 400 μg of BMP-2 (n = 1). The construct was loaded into a rigid bioabsorbable cage for implantation. Fusion rate and quality were assessed 2 months after operation. Manual palpation revealed successful fusion in all groups. Radiological fusion score of PEC groups was, however, higher than that of ACS groups. The newly formed bone in PEC groups appeared to be well integrated into the native bone with no overgrowth into the adjacent structure. On comparison, in ACS groups, large gaps were observed between the newly formed bone and the fusion bed with heterotopic ossification into the psoas muscle. The microarchitecture on the newly formed bone in PEC groups was superior to that in ACS groups, which was demonstrated by higher three-dimensional parameters. The present study demonstrated that BMP-2 delivered by PEC induced successful posterolateral fusion in porcine model. The efficacy of BMP-2 was improved and bony overgrowth was reduced. The microarchitecture of BMP-2-induced bone tissue was also enhanced by PEC. N/A.

Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats

Background ContextThe combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. PurposeWe hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. Study Design/SettingA prospective study using a rodent model of posterolateral spinal fusion was carried out. Patient SampleThirty-six syngeneic Fischer rats comprised the patient sample. MethodsSix groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. ResultsRat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and β-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). ConclusionIn summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct.

Dual Delivery of EPO and BMP2 from a Novel Modular Poly-ɛ-Caprolactone Construct to Increase the Bone Formation in Prefabricated Bone Flaps.

Poly-ɛ-caprolactone (PCL) is a biocompatible polymer that has mechanical properties suitable for bone tissue engineering; however, it must be integrated with biologics to stimulate bone formation. Bone morphogenetic protein-2 (BMP2) delivered from PCL produces bone when implanted subcutaneously, and erythropoietin (EPO) works synergistically with BMP2. In this study, EPO and BMP2 are adsorbed separately on two 3D-printed PCL scaffold modules that are assembled for codelivery on a single scaffold structure. This assembled modular PCL scaffold with dual BMP2 and EPO delivery was shown to increase bone growth in an ectopic location when compared with BMP2 delivery along a replicate scaffold structure. EPO (200 IU/mL) and BMP2 (65 μg/mL) were adsorbed onto the outer and inner portions of a modular scaffold, respectively. Protein binding and release studies were first quantified. Subsequently, EPO+BMP2 and BMP2 scaffolds were implanted subcutaneously in mice for 4 and 8 weeks, and the regenerated bone was analyzed with microcomputed tomography and histology; 8.6±1.4 μg BMP2 (22%) and 140±29 IU EPO (69.8%) bound to the scaffold and <1% BMP2 and 83% EPO was released in 7 days. Increased endothelial cell proliferation on EPO-adsorbed PCL discs indicated protein bioactivity. At 4 and 8 weeks, dual BMP2 and EPO delivery regenerated more bone (5.1±1.1 and 5.5±1.6 mm(3)) than BMP2 alone (3.8±1.1 and 4.3±1.7 mm(3)). BMP2 and EPO scaffolds had more ingrowth (1.4%±0.6%) in the outer module when compared with BMP2 (0.8%±0.3%) at 4 weeks. Dual delivery produced more dense cellular marrow, while BMP2 had more fatty marrow. Dual EPO and BMP2 delivery is a potential method to regenerate bone faster for prefabricated flaps.

Biodegradable polymer scaffolds loaded with low-dose BMP-2 stimulate periodontal ligament cell differentiation.

Poly(L-lactide)-co-(epsilon-caprolactone) [poly(LLA-co-CL)] and poly(L-lactide)-co-(1,5-dioxepan-2-one) [poly(LLA-co-DXO)] are being considered candidate scaffolds for bone tissue engineering. We evaluated the bioactive potential of poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds loaded with low-dose bone morphogenetic protein-2 (BMP-2). Periodontal ligament (PDL) cells were cultured onto the various scaffolds loaded with 1 µg BMP-2 or without BMP-2 (control). Cell viability, attachment, and proliferation were determined using a methylthiazol tetrazolium (MTT) colorimetric assay at day 1, 3, and 7. Scanning electron microscopy was used to analyze cell morphology at day 7. Cell differentiation was evaluated assaying alkaline phosphatase (ALP) activity at day 7, 14, and 21. Real-time PCR was used to evaluate the mRNA expression of periostin, ALP, type I collagen, bone sialoprotein and BMP-2. A commercially available enzyme-linked immunosorbent assay was used to assess BMP-2 production. Surface analysis disclosed excellent cell attachment, spread, and penetration into the porous scaffolds. The MTT assay indicated that scaffolds loaded with low concentration of BMP-2 did not influence the viability of cells. Cells grown on the modified scaffolds expressed higher levels of osteogenic markers than the nonmodified scaffolds (p<0.05). Poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds loaded with low-dose BMP-2 exhibited a significant effect stimulating PDL differentiation suggesting a continued evaluation in relevant in vivo models.

Enhancement of tendon-bone healing with the use of bone morphogenetic protein-2 inserted into the suture anchor hole in a rabbit patellar tendon model

Background aimsSuture anchor fixation failure has been reported as a result of anchor loosening and migration during the tendon-bone repair. The aim of this study was to evaluate the effects of bone morphogenetic protein-2 (BMP-2) inserted into the suture anchor hole on bone formation and the tendon-bone healing. MethodsBoth back legs of 24 New Zealand White rabbits (n = 48) were used in this study. A metal suture anchor was then placed 5 mm below the cortex. In the control group, the space over the eyelet of the anchor (suture anchor hole) was not filled. In the experimental group, the suture anchor hole was filled with 0.1 mL of fibrin glue (group 2) or collagen gel (group 3) with 1 μg BMP-2. Histologic analysis, real-time-polymerase chain reaction, bone density and failure load measurement were performed, and differences were analyzed at 4 and 8 weeks. ResultsHistologic analysis revealed more abundant new bone, mature bone and organized fibrocartilage at the tendon-bone interface at 4 and 8 weeks in groups in which BMP-2 was applied. At 8 weeks, the failure load of groups 1, 2 and 3 was significantly different among the three groups (P = 0.01). After post hoc Tukey test, the failure load of group 2 was significantly higher than that of group 1 (P = 0.01). ConclusionsBMP-2, administrated as described in this study, improved tendon-bone healing and bone formation, resulting in improved biomechanical strength of the tendon-bone junction.

Osteoinductivity of gelatin/β-tricalcium phosphate sponges loaded with different concentrations of mesenchymal stem cells and bone morphogenetic protein-2 in an equine bone defect model

Fracture is one of the most life-threatening injuries in horses. Fracture repair is often associated with unsatisfactory outcomes and is associated with a high incidence of complications. This study aimed to evaluate the osteogenic effects of gelatin/β-tricalcium phosphate (GT) sponges loaded with different concentrations/ratios of mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) in an equine bone defect model. Seven thoroughbred horses were used in this study. Eight bone defects were created in the third metatarsal bones of each horse. Then, eight treatments, namely control, GT, GT/M-5, GT/M-6, GT/M-5/B-1, GT/M-5/B-3, GT/M-6/B-1, and GT/M-6/B-3 were applied to the eight different sites in a randomized manner (M-5: 2 × 10(5) MSCs; M-6: 2 × 10(6) MSCs; B-1: 1 μg of BMP-2; B-3: 3 μg of BMP-2). Repair of bone defects was assessed by radiography, quantitative computed tomography (QCT), and histopathological evaluation. Radiographic scores and CT values were significantly lower in the control group than in the other groups, while they were significantly higher in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The amount of mature compact bone filling the defects was greater in the GT/M-5/B-3 and GT/M-6/B-3 groups than in the other groups. The present study demonstrated that the GT sponge loaded with MSCs and BMP-2 promoted bone regeneration in an equine bone defect model. The GT/MSC/BMP-2 described here may be useful for treating horses with bone injuries.

Cartilage repair by local delivery of transforming growth factor-β1 or bone morphogenetic protein-2 from a novel, segmented polyurethane/polylactic-co-glycolic bilayered scaffold

This study aimed to analyze the in vitro and in vivo release kinetics and evaluate the grades of repair induced by either the release of 50 ng of transforming growth factor-β1 or 2.5 or 5 μg of bone morphogenetic protein-2 (BMP-2) from a bilayer scaffold of segmented polyurethane/polylactic-co-glycolic (SPU/PLGA) in osteochondral defects, in a rabbit model. The scaffold consisted of a porous, bone-directed PLGA layer, overlaid with a cartilage-directed layer of growth factor (GF)-loaded PLGA microspheres, dispersed in a matrix of SPU. The PLGA porous layer was fabricated by gas foaming. Microspheres were prepared by a double emulsion method. SPU was synthesized by following the two-step method. GF release kinetics were assessed using iodinated ((125)I) GFs. The in vivo release profiles of both GFs fitted to zero-order kinetics, demonstrating a consistently good control of their release rates by SPU. Cartilage-like tissue, characterized by histological analysis, scoring, and immunolabeling of chondrogenic differentiation markers, was observed only after 12 weeks, maintaining integrity up to at least 24 weeks, independently of the GF and the dose of BMP-2. The biocompatibility and the resulting good quality, hyaline repair cartilage convert this system into a promising candidate for future applications in osteochondral lesions.