Release Kinetics Of Bone Morphogenetic Protein-2 Research Articles

Sustained BMP-2 delivery via alginate microbeads and polydopamine-coated 3D-Printed PCL/β-TCP scaffold enhances bone regeneration in long bone segmental defects

Background/ObjectiveRepair of long bone defects remains a major challenge in clinical practice, necessitating the use of bone grafts, growth factors, and mechanical stability. Hence, a combination therapy involving a 3D-printed polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP) scaffold coated with polydopamine (PDA) and alginate microbeads (AM) for sustained delivery of bone morphogenetic protein-2 (BMP-2) was investigated to treat long bone segmental defects. MethodsSeveral in vitro analyses were performed to evaluate the scaffold osteogenic effects in vitro such as PDA surface modification, namely, hydrophilicity and cell adhesion; cytotoxicity and BMP-2 release kinetics using CCK-8 assay and ELISA, respectively; osteogenic differentiation in canine adipose-derived mesenchymal stem cells (Ad-MSCs); formation of mineralized nodules using ALP staining and ARS staining; and mRNA expression of osteogenic differentiation markers using RT-qPCR. Bone regeneration in femoral bone defects was evaluated in vivo using a rabbit femoral segmental bone defect model by performing radiography, micro-computed tomography, and histological observation (hematoxylin and eosin and Masson's trichrome staining). ResultsThe PDA-coated 3D-printed scaffold demonstrated increased hydrophilicity, cell adhesion, and cell proliferation compared with that of the control. BMP-2 release kinetics assessment showed that BMP-2 AM showed a reduced initial burst and continuous release for 28 days. In vitro co-culture with canine Ad-MSCs showed an increase in mineralization and mRNA expression of osteogenic markers in the BMP-2 AM group compared with that of the BMP-2-adsorbed scaffold group. In vivo bone regeneration evaluation 12 weeks after surgery showed that the BMP-2 AM/PDA group exhibited the highest bone volume in the scaffold, followed by the BMP-2/PDA group. High cortical bone connectivity was observed in the PDA-coated scaffold groups. ConclusionThese findings suggest that the combined use of PDA-coated 3D-printed bone scaffolds and BMP-2 AM can successfully induce bone regeneration even in load-bearing bone segmental defects. The translational potential of this articleA 3D-printed PCL/β-TCP scaffold was fabricated to mimic the cortical bone of the femur. Along with the application of PDA surface modification and sustained BMP-2 release via AM, the developed scaffold could provide suitable osteoconduction, osteoinduction, and osteogenesis in both in vitro settings and in vivo rabbit femoral segmental bone defect models. Therefore, our findings suggest a promising therapeutic option for treating challenging long bone segmental defects, with potential for future clinical application.

Layer-by-layer assembly of poly-l-lysine/hyaluronic acid protein reservoirs on poly(glycerol sebacate) surfaces

The modification of biomaterial surfaces has become increasingly relevant in the context of ongoing advancements in tissue engineering applications and the development of tissue-mimicking polymer materials. In this study, we investigated the layer-by-layer (LbL) deposition of polyelectrolyte multilayer protein reservoirs consisting of poly-l-lysine (PLL) and hyaluronic acid (HA) on the hydrophobic surface of poly(glycerol sebacate) (PGS) elastomer. Using the methods of isothermal titration calorimetry and surface plasmon resonance, we systematically investigated the interactions between the polyelectrolytes and evaluated the deposition process in real time, providing insight into the phenomena associated with film assembly. PLL/HA LbL films deposited on PGS showed an exceptional ability to incorporate bone morphogenetic protein-2 (BMP-2) compared to other growth factors tested, thus highlighting the potential of PLL/HA LbL films for osteoregenerative applications. The concentration of HA solution used for film assembly did not affect the thickness and topography of the (PLL/HA)10 films, but had a notable impact on the hydrophilicity of the PGS surface and the BMP-2 release kinetics. The release kinetics were successfully described using the Weibull model and hyperbolic tangent function, underscoring the potential of these less frequently used models to compare the protein release from LbL protein reservoirs.

Effectiveness of BMP-2 and PDGF-BB Adsorption onto a Collagen/Collagen-Magnesium-Hydroxyapatite Scaffold in Weight-Bearing and Non-Weight-Bearing Osteochondral Defect Bone Repair: In Vitro, Ex Vivo and In Vivo Evaluation

Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.

Hybrid Implants Based on Calcium-Magnesium Silicate Ceramics Diopside as a Carrier of Recombinant BMP-2 and Demineralized Bone Matrix as a Scaffold: Dynamics of Reparative Osteogenesis in a Mouse Craniotomy Model.

Calcium-magnesium silicate ceramics, diopside, is a promising material for use in bone plastics, but until now the possibility of its use as a carrier of recombinant bone morphogenetic protein-2 (BMP-2) has not been studied, as well as the features of reparative osteogenesis mediated by the materials based on diopside with BMP-2. Powder of calcium-magnesium silicate ceramics was obtained by solid-state synthesis using biowaste - rice husks and egg shells - as source components. Main phase of the obtained ceramics was diopside. The obtained particles were irregularly shaped with an average size of about 2.3 μm and ~20% porosity; average pore size was about 24 nm, which allowed the material to be classified as mesoporous. Diopside powder adsorbs more than 150 μg of recombinant BMP-2 per milligram, which exceeds binding capacity of hydroxyapatite, a calcium-phosphate ceramic often used in hybrid implants, by more than 3 times. In vitro release kinetics of BMP-2 was characterized by a burst release in the first 2 days and a sustained release of approximately 0.4 to 0.5% of the loaded protein over the following 7 days. In vivo experiments were performed with a mouse model of cranial defects of critical size with implantation of a suspension of diopside powder with/without BMP-2 in hyaluronic acid incorporated into the disks of demineralized bone matrix with 73-90% volume porosity and macropore size from 50 to 650 μm. Dynamics of neoosteogenesis and bone tissue remodeling was investigated histologically at the time points of 12, 21, 48, and 63 days. Diopside particles were evenly spread in the matrix and caused minimal foreign body reaction. In the presence of BMP-2 by the day 63 significant foci of newly formed bone tissue were formed in the implant pores with bone marrow areas, moreover, large areas of demineralized bone matrix in the implant center and maternal bone at the edges were involved in the remodeling. Diopside could be considered as a promising material for introduction into hybrid implants as an effective carrier of BMP-2.

8. Controlling the rate of presentation of BMP-2 from a highly structured composite for interbody fusion

<h3>BACKGROUND CONTEXT</h3> Clinical outcomes for patients with intervertebral disc injury or disease could be enhanced by a new generation of regenerative matrices that create tissue environments to control angiogenesis, chondrogenesis and bone formation. Regenerative matrices are a new class of controlled release biomaterials that use surface chemistry, materials architecture, and drug delivery kinetics to create optimised conditions for cell interactions that result in high quality tissue formation. A novel biodegradable polymer matrix that releases bone morphogenetic protein 2 (BMP-2) over a period of one month with a low Cmax and sustained presentation of active protein is investigated in this study. <h3>PURPOSE</h3> BMP-2 release kinetics and protein activity from a new regenerative matrix was quantified to develop an implant with 1-month release of the drug within a macroporous matrix. A rabbit posterolateral (PLF) vertebral fusion model was performed to assess bone formation and fusion outcomes as a function of dose and source of BMP-2 (E coli and CHO cells). <h3>STUDY DESIGN/SETTING</h3> The study was designed to investigate the effect of various biomaterial design factors and manufacturing methods on the rate of release of BMP-2, the activity of BMP-2 and the extent of in vivo bone formation. <h3>PATIENT SAMPLE</h3> This study did not involve patients. <h3>OUTCOME MEASURES</h3> Outcome measures were the mechanical properties of the regenerative matrix, the architecture and porosity of the matrix, BMP-2 release kinetics, BMP-2 activity as a function of release time and bone formation. <h3>METHODS</h3> BMP-2 activity was tested using two in vitro cell-based assays. BMP-2 solutions and regenerative matrix extracts were tested for their ability to induce alkaline phosphatase (ALP) activity in W-20-17 stromal cells using a modified version of the ASTM standard protocol for in vitro biological activity of recombinant human BMP-2 (F2131-02). Cells were exposed to serial dilutions of BMP-2 for 24 h, assayed for ALP activity and EC50s calculated allowing the relative potency to NIBSC WHO BMP-2 standard to be calculated. BMP-2 release was assessed with an in vitro assay of ALP activity in the mouse muscle myoblast cell line C2C12. Formulations were suspended above a C2C12 monolayer in an insert with a porous (8 µm) PET membrane. Following 3-4 days of exposure, the inserts containing LDGraft were transferred to fresh cells for a further 3-4 days. Cells exposed to LDGraft in this manner were assayed for ALP activity (normalised to protein content) once the inserts had been transferred to fresh cells. Fusion rate and new bone formation were compared to INFUSE in a single-level lumbar (L4-L5) posterolateral (PLF) vertebral fusion model in the New Zealand White rabbit based on standard protocols (Boden et al, 1995, ASTM F3207). Pre-formed implants (3 cc) of INFUSE (rhBMP-2 and absorbable collagen sponge) were implanted on decorticated dorsal surfaces of the transverse processes of L4 and L5 adjacent to the laminae. Fusion was assessed by radiograph at 6 weeks and by manual palpation, radiography and microCT at 12 weeks. <h3>RESULTS</h3> BMP-2 activity was retained over one month of release with the addition of protectants in the formulation and precise control of manufacturing processes. The matrix had a porosity of >70% with a high proportion of macropores (>200 micron diameter). Low doses of E. coli-produced BMP-2 induced fusion in the PLF model. <h3>CONCLUSIONS</h3> A one-month releasing BMP-2 formulation is described. This new regenerative matrix had internal architecture designed to host angiogenesis. Manufacturing and design challenges can be overcome to retain high biologic activity throughout the release period. <h3>FDA DEVICE/DRUG STATUS</h3> BMP-2, not approved for this indication.

Sustained release of BMP-2 using self-assembled layer-by-layer film-coated implants enhances bone regeneration over burst release

Current clinical products delivering the osteogenic growth factor bone morphogenetic protein 2 (BMP-2) for bone regeneration have been plagued by safety concerns due to a high incidence of off-target effects resulting from bolus release and supraphysiological doses. Layer-by-layer (LbL) film deposition offers the opportunity to coat bone defect-relevant substrates with thin films containing proteins and other therapeutics; however, control of release kinetics is often hampered by interlayer diffusion of drugs throughout the film during assembly, which causes burst drug release. In this work, we present the design of different laponite clay diffusional barrier layer architectures in self-assembled LbL films to modulate the release kinetics of BMP-2 from the surface of a biodegradable implant. Release kinetics were tuned by incorporating laponite in different film arrangements and with varying deposition techniques to achieve release of BMP-2 over 2 days, 4 days, 14 days, and 30 days. Delivery of a low dose (0.5 μg) of BMP-2 over 2 days and 30 days using these LbL film architectures was then compared in an in vivo rat critical size calvarial defect model to determine the effect of BMP-2 release kinetics on bone regeneration. After 6 weeks, sustained release of BMP-2 over 30 days induced 3.7 times higher bone volume and 7.4 times higher bone mineral density as compared with 2-day release of BMP-2, which did not induce more bone growth than the uncoated scaffold control. These findings represent a crucial step in the understanding of how BMP-2 release kinetics influence treatment efficacy and underscore the necessity to optimize protein delivery methods in clinical formulations for bone regeneration. This work could be applied to the delivery of other therapeutic proteins for which careful tuning of the release rate is a key optimization parameter.

Shape-Memory ECM-Mimicking Heparin-Modified Nanofibrous Gelatin Scaffold for Enhanced Bone Regeneration in Sinus Augmentation.

Biomaterials with clinical maneuverability and predictable bone regeneration are needed in the field of maxillary sinus augmentation. Herein, gelatin was chemically modified with heparin that specifically interacted with and stabilized bone morphogenetic protein-2 (BMP-2). We then introduced thermally induced phase separation to form the injectable, shape-memory, highly porous scaffold for bone regeneration in sinus augmentation. The hydrated heparin-modified nanofibrous gelatin scaffolds (NH-GS) were demonstrated with high resilience and shape-memory property, both macroscopically and microscopically, making them injectable scaffolds and expected to be applied in sinus augmentation. This novel scaffold was verified to be biocompatible and an excellent matrix to support cell attachment, proliferation, and infiltration. Further, the growth factor-loaded NH-GS showed sustained release kinetics of BMP-2 through affinity-based scaffold-growth factor interaction, compared with BMP-2 loaded gelatin sponge (GS) and nanofibrous gelatin scaffold (NF). Both in vitro and in vivo experiments demonstrated that the BMP-2-loaded NH-GS exhibited the highest osteogenesis among the other groups. Taken together, this study introduces a new regenerative strategy in maxillary sinus augmentation, which is injectable with a predefined shape and structure and promotes bone regeneration through a more sustained BMP-2 release.

A novel nano-hydroxyapatite/synthetic polymer/bone morphogenetic protein-2 composite for efficient bone regeneration

BACKGROUNDEfficient bone regeneration using recombinant human bone morphogenetic protein-2 (BMP-2) is needed to reduce side effects caused by high-dose BMP-2 use. The composite material of polylactic acid–polyethene glycol (PLA-PEG) for sustained release and an osteogenic nano-hydroxyapatite (nHAp) can contribute to efficient bone regeneration by BMP-2. STUDY DESIGNAn experimental in vitro and in vivo study. PURPOSEThe objective of this study is to investigate the effectiveness of a novel composite material of PLA-PEG and nHAp as a carrier for BMP-2. METHODSThe release kinetics of BMP-2 from the composites was investigated by ELISA. Thirty-six male Sprague–Dawley rats underwent posterolateral spinal fusion on L4–L5 with three different doses of BMP-2 (0 µg [control], 3 µg [low dose], and 10 µg [high dose]). Weekly µCT results and histology and a manual palpation test at 8 weeks postoperatively were used for assessment of the spinal fusion. RESULTSELISA demonstrated the sustained release of BMP-2 until day 21. µCT and manual palpation test demonstrated a solid fusion in 91.6% (11/12) of specimens in both the low- and high-dose groups. N mice in the control group attained bony fusion (0%, 0/9). nHAp was resorbed between 2 and 4 weeks postoperatively, and regenerated fusion mass at 8 weeks postoperatively consisted of only newly formed bone. CONCLUSIONSThe nHAp/PLA-PEG composite enabled efficient bone regeneration with low-dose BMP-2. The sustained release of BMP-2 by PLA-PEG and the osteogenic and biodegradable scaffold of nHAp might contribute to efficient bone regeneration. CLINICAL SIGNIFICANCEThis novel composite material has potential in clinical applications (spinal fusion, large bone defect and non-union) by enabling efficient bone formation by BMP-2.

Core Ossification of Bone Morphogenetic Protein-2-Loaded Collagenated Bone Mineral in the Sinus.

The objective of this study was to investigate in vitro release kinetics and ossification patterns of bone morphogenetic protein-2-soaked collagenated porcine bone mineral (BMP-2/CPBM) in rabbit sinuses. Release kinetics of BMP-2/CPBM was determined in vitro up to 56 days. In 16 rabbits, BMP-2/CPBM (BMP group) and CPBM alone (control group) were bilaterally grafted in both sinuses. After 4 (N = 8) and 12 (N = 8) weeks, radiographic and histologic analyses were performed. Approximately 40% of BMP-2 was released from CPBM during 3 days in vitro; release maintained at a reduced level until day 56. In vivo, new bone formation in BMP group was dominant at the center and decreased toward the borders of the sinus, while it mainly possessed close to the sinus membrane and basal bone in control group. At the center, significantly more new bone was found in BMP group compared to control group at 4 weeks (29.14% vs. 16.50%; p < 0.05). The total augmented volume of BMP group was significantly greater than control group at 4 (370.13 mm3 vs. 299.32 mm3) and 12 (400.40 mm3 vs. 290.10 mm3) weeks (p < 0.05). In conclusion, BMP-2/CPBM demonstrated a core ossification with a greater augmented volume and new bone formation in the center of the sinus compared to CPBM alone. Impact statement The center of the augmented maxillary sinus tends to show a slower and inferior new bone formation compared to the sites near the sinus membrane and basal bone. In this study, bone morphogenetic protein-2 (BMP-2) loaded onto collagenated porcine bone mineral (CPBM) resulted in a greater augmented volume and new bone formation at the center of the grafted sinus compared to CPBM alone. Therefore, BMP-2-added CPBM in maxillary sinus augmentation may potentially be beneficial to the clinicians, in terms of accelerating the new bone formation at the center area where the apical half of the implant fixture usually places.

Electrospun Fibers Immobilized with BMP-2 Mediated by Polydopamine Combined with Autogenous Tendon to Repair Developmental Dysplasia of the Hip in a Porcine Model.

PurposeDevelopmental dysplasia of the hip (DDH) can increase the pressure between the joints, which causes secondary hip osteoarthritis. The aim of the present study was to fabricate poly(D, L-lactic acid)-poly(ethylene glycol)-poly(D, L-lactic acid) (PELA) electrospun fibrous scaffolds, immobilized with bone morphogenetic protein-2 (BMP-2), to repair the acetabulum defects.MethodsThe characteristics of PELA electrospun were analyzed using scanning electron microscopy, the release kinetics of BMP-2 in vitro were analyzed using enzyme-linked immunosorbent assays. Human mesenchymal stem cells (hMSCs) were used for in vitro experiments, the biocompatibility of the electrospinning materials was investigated using a cell counting kit-8 (CCK-8) kit, and osteogenic differentiation was tested via alkaline phosphatase (ALP) activity and relative gene expression. Eighteen miniature pig animal models were used in the in vivo experiment. The pigs were sacrificed at 24 weeks after surgery, and the reconstructed acetabulum was evaluated using histological sections.ResultsStructural analysis revealed that the diameter of the PELA electrospun fiber was 0.8195 ± 0.16 μm. The PELA electrospun fiber materials showed good hydrophilicity and biocompatibility and could continuously release BMP-2 within 27 days: 16.07 ± 0.11 ng of BMP-2 was released from the scaffold. A total of sixteen implants fully filled the defects, and hematoxylin and eosin staining and Goldner’s trichrome staining showed that the simple tendon group (T group) was mostly fibrous tissues, lots of fibroblasts and small amounts of chondrocytes were observed in the polydopamine-coated electrospun fiber group (DP group). The DP plus BMP-2 (DPB) group showed a large number of chondrocytes and partial new bone tissues.ConclusionPELA electrospun fibrous scaffolds are good sustained-release carriers, which can not only induce implant differentiation into cartilage and bone but also are completely degraded without toxicity. Therefore, the material can be used for bone and cartilage regeneration.

Comparison of Impregnated Bone Morphogenetic Protein-2 Release Kinetics from Biopolymer Scaffolds

The purpose of this study was to evaluate the release kinetics of impregnated recombinant human bone morphogenetic protein-2 (rhBMP-2) from different engineered scaffolds. Poly(lactide-co-glycolide) (PLG) matrices prepared by supercritical fluid technologies (SCFT) showed the highest biocompatibility and long-term release of rhBMP-2. There was an even release of rhBMP-2 from them for 11 days. The subsequent use of laser sintering allowed delaying the peak of the protein release for a period of 13 to 15 days. The average loss of rhBMP-2 using SCFT did not exceed 20%. The maximum release of rhBMP-2 from a collagen-fibronectin hydrogel was in the period from 4 to 6 days. But 47 ± 12% rhBMP-2 loss was shown. Highly porous polylactide-based scaffolds obtained by freeze-drying were inferior to other scaffolds in their ability to release rhBMP-2 for a prolonged period. The hydrogel and chitosan-based granules showed high cytotoxicity and a short period of protein release.

Сравнение кинетики высвобождения импрегнированного костного морфогенетического белка-2 из биополимерных матриксов

Сравнение кинетики высвобождения импрегнированного костного морфогенетического белка-2 из биополимерных матриксов

Enhanced Skull Bone Regeneration by Sustained Release of BMP-2 in Interpenetrating Composite Hydrogels.

Direct administration of bone morphogenetic protein-2 (BMP-2) for bone regeneration could cause various clinical side effects such as osteoclast activation, inflammation, adipogenesis, and bone cyst formation. In this study, thiolated gelatin/poly(ethylene glycol) diacrylate (PEGDA) interpenetrating (IPN) composite hydrogels were developed for guided skull bone regeneration. To promote bone regeneration, either polycation-based coacervates (Coa) or gelatin microparticles (GMPs) were incorporated within IPN gels as BMP-2 carriers. Both BMP-2 loaded Coa and BMP-2 loaded GMPs showed significantly enhanced in vitro alkaline phosphate (ALP) activity of human mesenchymal stem cells (hMSCs) than non-BMP-2 treated control. Moreover, BMP-2 loaded GMPs group exhibited statistically increased ALP activity compared to both bolus BMP-2 administration and BMP-2 loaded Coa group, indicating that our carriers could protect and maintain biological activity of cargo BMP-2. Sustained release kinetics of BMP-2 from IPN composite hydrogels could be controlled by different formulations. For in vivo bone regeneration, various IPN gel formulations (i.e., (1) control, (2) only hydrogel, (3) hydrogel with bolus BMP-2, (4) hydrogel with BMP-2-loaded Coa, and (5) hydrogel with BMP-2-loaded GMPs) were bilaterally implanted into 5 mm-sized rat calvarial defects. After 4 weeks, micro-CT and histological analysis were performed to evaluate new bone formation. Significantly higher scores for bony bridging and union were observed in BMP-2-loaded Coa and BMP-2-loaded GMP groups as compared to other formulations. In addition, rats treated with BMP-2-loaded GMPs showed a significantly higher ratio of bone volume/total volume and lower trabecular separation scores than others. Finally, rats treated with either Coa or GMP groups exhibited a significant increase in bone formation area, as assessed via histomorphometric analysis. Taken together, it could be concluded that Coa and GMPs were effective carriers to maintain the bioactivity of cargo BMP-2 during its sustained release. Consequently, our IPN composite hydrogel system that combines such BMP-2 carriers could effectively promote skull bone regeneration.

Application of alginate microbeads as a carrier of bone morphogenetic protein-2 for bone regeneration.

Bone morphogenetic protein-2 (BMP-2) is commonly used to enhance bone regeneration. The potential of BMP-2 for bone regeneration varies according to the concentration and release kinetics on the implanted site. Therefore, it is important to determine appropriate carriers of BMP-2. However, no optimal delivery vehicles have been identified. In the present study, we used alginate microbeads as a delivery vehicle for BMP-2. Alginate microbeads can be implanted onto the disease site through surgery or injection. The objective of this study was to evaluate that the osteoinductive properties of BMP-2 are effective in alginate microbeads as a carrier. In this study, the release kinetics of BMP-2 in alginate microbeads was evaluated using an enzyme-linked immunosorbent assay. BMP-2 released from alginate microbeads induced high alkaline phosphatase activity in canine adipose tissue-derived mesenchymal stem cells. Injection of alginate microbeads with BMP-2 into mouse subcutaneous tissue, as well as surgical implantation into the 5-mm circular calvarial defects in rats, was conducted and the results showed extensive new bone formation. In conclusion, alginate microbeads can be utilized as an effective BMP-2 delivery vehicle for use in orthopedic surgery and as an injectable vehicle for a minimally invasive therapy. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 286-294, 2019.

Combined sustained release of BMP2 and MMP10 accelerates bone formation and mineralization of calvaria critical size defect in mice

The effect of dual delivery of bone morphogenetic protein-2 (BMP-2) and matrix metalloproteinase 10 (MMP10) on bone regeneration was investigated in a murine model of calvarial critical-size defect, hypothesizing that it would result in an enhanced bone formation. Critical-size calvarial defects (4 mm diameter) were created in mice and PLGA microspheres preloaded with either BMP-2, MMP10 or a microsphere combination of both were transplanted into defect sites at different doses. Empty microspheres were used as the negative control. Encapsulation efficiency was assessed and in vivo release kinetics of BMP-2 and MMP10 were examined over 14 days. Histological analyses were used to analyze bone formation after four and eight weeks. Combination with MMP10 (30 ng) significantly enhanced BMP-2 (600 ng)-mediated osteogenesis, as confirmed by the increase in percentage of bone fill (p < .05) at four weeks. Moreover, it also increased mineral apposition rate (p < .05), measured by double labeling with tetracycline and calceine. MMP10 accelerates bone repair by enhancing BMP-2-promoted bone healing and improving the mineralization rate. In conclusion combination of MMP10 and BMP-2 may become a promising strategy for repair and regeneration of bone defects.

Collagen I derived recombinant protein microspheres as novel delivery vehicles for bone morphogenetic protein-2

Bone morphogenetic protein-2 (BMP-2) is a powerful osteoinductive protein; however, there is a need for the development of a safe and efficient BMP-2 release system for bone regeneration therapies. Recombinant extracellular matrix proteins are promising next generation biomaterials since the proteins are well-defined, reproducible and can be tailored for specific applications. In this study, we have developed a novel and versatile BMP-2 delivery system using microspheres from a recombinant protein based on human collagen I (RCP). In general, a two-phase release pattern was observed while the majority of BMP-2 was retained in the microspheres for at least two weeks. Among different parameters studied, the crosslinking and the size of the RCP microspheres changed the in vitro BMP-2 release kinetics significantly. Increasing the chemical crosslinking (hexamethylene diisocyanide) degree decreased the amount of initial burst release (24h) from 23% to 17%. Crosslinking by dehydrothermal treatment further decreased the burst release to 11%. Interestingly, the 50 and 72μm-sized spheres showed a significant decrease in the burst release compared to 207-μm sized spheres. Very importantly, using a reporter cell line, the released BMP-2 was shown to be bioactive. SPR data showed that N-terminal sequence of BMP-2 was important for the binding and retention of BMP-2 and suggested the presence of a specific binding epitope on RCP (KD: 1.2nM). This study demonstrated that the presented RCP microspheres are promising versatile BMP-2 delivery vehicles.

Effect of different sustained bone morphogenetic protein‐2 release kinetics on bone formation in poly(propylene fumarate) scaffolds

To investigate the effect of sustained bone morphogenetic protein-2 (BMP-2) release kinetics on bone formation in poly(propylene fumarate) (PPF) scaffolds, different poly(lactic-co-glycolic acid) (PLGA) microspheres were used as delivery vehicles. All PPF scaffolds had the same 75% porous structure, while the degradation rate of the embedded PLGA microspheres was changed to tailor BMP-2 release by varying the lactic-to-glycolic acid (L:G) ratio in the copolymer. Four PLGA microsphere formulations with 50/50, 65/35, 75/25, and 85/15 L:G ratios and varying in vivo degradation rates were fabricated. The in vitro and in vivo BMP-2 release kinetics were determined by analyzing radiolabeled 125 I-BMP-2. Biological activity of released BMP-2 was tested using a W20-17 cell culture model in vitro and a subcutaneous rat model in vivo. Corresponding outcome parameters were defined as capacity to increase the in vitro AP activity in weekly consecutive cell cultures over 14 weeks and the in vivo bone formation after 7 and 14 weeks. The PLGA/PPF composites showed similar biological activity and BMP-2 release profiles in vitro. In vivo, PPF/PLGA 85:15 composite released significantly less BMP-2 per time point in the first weeks. Micro-CT and histological analysis after 7 and 14 weeks of implantation showed bone formation, which significantly increased over time for all composites. No significant differences were seen between the composites. Overall, the results of this study show that small differences in BMP-2 sustained release had no significant effect on BMP-2 osteogenic efficacy in PPF/PLGA composites. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 477-487, 2018.

Bone regeneration in critical-sized bone defect enhanced by introducing osteoinductivity to biphasic calcium phosphate granules.

Biphasic calcium phosphate (BCP) is frequently used as bone substitute and often needs to be combined with autologous bone to gain an osteoinductive property for guided bone regeneration in implant dentistry. Given the limitations of using autologous bone, bone morphogenetic protein-2 (BMP2)-coprecipitated, layer-by-layer assembled biomimetic calcium phosphate particles (BMP2-cop.BioCaP) have been developed as a potential osteoinducer. In this study, we hypothesized that BMP2-cop.BioCaP could introduce osteoinductivity to BCP and so could function as effectively as autologous bone for the repair of a critical-sized bone defect. We prepared BMP2-cop.BioCaP and monitored the loading and release kinetics of BMP2 from it invitro. Seven groups (n=6 animals/group) were established: (i) Empty defect; (ii) BCP; (iii) BCP mixed with biomimetic calcium phosphate particles (BioCaP); (iv) BCP mixed with BMP2-cop.BioCaP; (v) BioCaP; (vi) BMP2-cop.BioCaP; (vii) BCP mixed with autologous bone. They were implanted into 8-mm-diameter rat cranial critical-sized bone defects for an invivo evaluation. Autologous bone served as a positive control. The osteoinductive efficacy and degradability of materials were evaluated using micro-CT, histology and histomorphometry. The combined application of BCP and BMP2-cop.BioCaP resulted in significantly more new bone formation than BCP alone. The osteoinductive efficacy of BMP2-cop.BioCaP was comparable to the golden standard use of autologous bone. Compared with BCP alone, significantly more BCP degradation was found when mixed with BMP2-cop.BioCaP. The combination of BCP and BMP2-cop.BioCaP showed a promising potential for guided bone regeneration clinically in the future.

Addition of a Synthetically Fabricated Osteoinductive Biphasic Calcium Phosphate Bone Graft to BMP2 Improves New Bone Formation.

Bone morphogenetic protein-2 (BMP2) has been successfully utilized in dentistry to promote new bone formation because of its osteoinductive ability to recruit mesenchymal progenitor cells and induce their differentiation to bone-forming osteoblasts. Recently, novel biphasic calcium phosphate scaffolds have been developed with similar osteoinductive properties capable of forming ectopic bone formation. The aim of the present study was to assess whether the combination of BMP2 with this novel Biphasic Calcium Phosphate (BCP) scaffold may additionally promote new bone regeneration. Cylindrical bone defects measuring 2.5 mm were created bilaterally in the femurs of 18 Wistar rats. After 4 weeks, the following six groups were assessed for new bone formation by micro-computed tomography (CT) as well as histological assessment: 1) collagen scaffolds + 20 μg of BMP2; 2) collagen scaffolds + 50 μg of BMP2; 3) collagen scaffolds + 100 μg of BMP2; 4) BCP scaffolds + 20 μg of BMP2; 5) BCP scaffolds + 50 μg of BMP2; and 6) BCP scaffolds + 100 μg of BMP2. Furthermore, tartrate-resistant acid phosphatase (TRAP) staining was utilized to assess osteoclast activity and osteoclast number. The release kinetics of BMP2 from both BCP and collagen scaffolds was investigated over a 14-day period. The results from present study demonstrate that BMP2 is able to promote new bone formation in a concentration dependant manner when loaded with either a collagen scaffolds or BCP scaffolds. Micro-CT analysis demonstrated significantly higher levels of new bone formation in groups containing BCP + BMP2 when compared with collagen scaffolds + BMP2. BMP2 had little effect on osteoclast activity; however, less TRAP staining and osteoclast number was observed in the defects receiving collagen scaffolds when compared with BCP scaffolds. The release of BMP2 over time was rapidly released after 1 day on BCP scaffolds whereas a gradually release over time was observed for collagen scaffolds up to 14 days. The osteoinductive properties of BMP2 may further be enhanced by its combination with a novel synthetically fabricated osteoinductive BCP scaffold. Future clinical testing is required to further assess these preliminary findings.

Novel osteoinductive photo-cross-linkable chitosan-lactide-fibrinogen hydrogels enhance bone regeneration in critical size segmental bone defects

The purpose of this study was to develop and characterize a novel photo-cross-linkable chitosan-lactide-fibrinogen (CLF) hydrogel and evaluate the efficacy of bone morphogenetic protein-2 (BMP-2) containing a CLF hydrogel for osteogenesis in vitro and in vivo. We synthesized the CLF hydrogels and characterized their chemical structure, degradation rate, compressive modulus and in vitro BMP-2 release kinetics. We evaluated bioactivities of the BMP-2 containing CLF hydrogels (0, 50, 100 and 500ngml−1) in vitro using W-20-17 preosteoblast mouse bone marrow stromal cells and C2C12 mouse myoblast cells. The effect of BMP-2 containing CLF gels (0, 0.5, 1, 2 and 5μg) on bone formation was evaluated using rat critical size segmental bone defects for 4weeks. Fourier transform infrared spectroscopy spectra and scanning electron microscopy images showed chemical and structural changes by the addition of fibrinogen into the chitosan-lactide copolymer. The incorporation of fibrinogen molecules significantly increased the compressive modulus of the hydrogels. The in vitro BMP-2 release study showed initial burst releases from the CLF hydrogels followed by sustained releases, regardless of the concentration of the BMP-2 over 4weeks. Cells in all groups were viable in the presence of the hydrogels regardless of BMP-2 doses, indicating non-cytotoxicity of hydrogels. Alkaline phosphate activity and mineralization of cells exhibited dose dependence on BMP-2 containing CLF hydrogels. Radiography, microcomputed tomography and histology confirmed that the BMP-2 containing CLF hydrogels prompted neo-osteogenesis and accelerated healing of the defects in a dose-dependent manner. Thus the CLF hydrogel is a promising delivery system of growth factors for bone regeneration.