Sustained Release Of BMP-2 Research Articles

REVITALIZATION OF CRITICAL-SIZED CALVARIAL DEFECTS IN RATS USING HEPARIN-CONJUGATED FIBRIN HYDROGEL WITH BMP-2 AND ADIPOSE-DERIVED PERICYTES

Massive bone defects present challenges in orthopedic surgery, requiring innovative solutions. Hydrogels offer promise due to their resemblance to the extracellular matrix. Adipose tissue-derived pericytes (ADPs) and osteoinductive proteins like BMP-2 show potential for bone tissue engineering. Combining them via heparin-conjugated fibrin hydrogel aims to enhance healing in critical-sized calvarial defects. The study investigated the use of a heparin-conjugated fibrin (HCF) hydrogel containing BMP-2 and adipose-derived pericytes for repairing critical-sized calvarial defects in rats. ADPs were cultured from rat adipose tissue, and the HCF hydrogel was prepared accordingly. In vitro experiments assessed BMP-2 release kinetics and bioactivity, while mineralization assays were conducted on cultured ADPs. In vivo experiments involved surgically creating calvarial defects in rats and implanting HCF gel alone or with BMP-2, pericytes, or both. Micro-CT imaging and histological analysis were performed post-implantation to evaluate bone formation. In our study, we prepared the HCF hydrogel by combining heparin-conjugated fibrinogen, human fibrinogen, human thrombin, aprotinin, and calcium chloride. Gelation occurred within 3 minutes at room temperature. SEM analysis revealed an open interconnected pore morphology and macroporous structure, facilitating cell attachment and proliferation. ELISA showed sustained release of BMP-2 from the HCF hydrogel over 28 days. Bioactivity assays demonstrated the ability of released BMP-2 to enhance ALP activity in rat neonatal calvarial osteoblasts. Additionally, BMP-2 induced osteogenic differentiation of rat ADPs, as evidenced by increased ALP activity, osteocalcin expression, and calcium deposition. Implantation of HCF hydrogels containing BMP-2 and/or ADPs into rat calvarial defects significantly promoted bone tissue regeneration compared to controls, with the greatest effect observed in the group receiving both BMP-2 and ADPs. Micro-CT and histological analysis confirmed substantial bone formation and defect closure after three months. In conclusion, our in vitro findings demonstrated that the HCF hydrogel provided sustained release of BMP-2, retaining its bioactivity and promoting osteogenesis in rat calvarial osteoblasts and ADPs. In vivo results showed that combining allogeneic ADPs with BMP-2 enhanced bone regeneration in critical-sized calvarial defects, indicating the potential of this approach for large bone defect restoration.

Polydopamine-mediated immobilization of BMP-2 onto electrospun nanofibers enhances bone regeneration

Dealing with bone defects is a significant challenge to global health. Electrospinning in bone tissue engineering has emerged as a solution to this problem. In this study, we designed a PVDF-b-PTFE block copolymer by incorporating TFE, which induced a phase shift in PVDF from α to β, thereby enhancing the piezoelectric effect. Utilizing the electrospinning process, we not only converted the material into a film with a significant surface area and high porosity but also intensified the piezoelectric effect. Then we used polydopamine to immobilize BMP-2 onto PVDF-b-PTFE electrospun nanofibrous membranes, achieving a controlled release of BMP-2. The scaffold’s characters were examined using SEM and XRD. To assess its osteogenic effects in vitro, we monitored the proliferation of MC3T3-E1 cells on the fibers, conducted ARS staining, and measured the expression of osteogenic genes. In vivo, bone regeneration effects were analyzed through micro-CT scanning and HE staining. ELISA assays confirmed that the sustained release of BMP-2 can be maintained for at least 28 d. SEM images and CCK-8 results demonstrated enhanced cell viability and improved adhesion in the experimental group. Furthermore, the experimental group exhibited more calcium nodules and higher expression levels of osteogenic genes, including COL-I, OCN, and RUNX2. HE staining and micro-CT scans revealed enhanced bone tissue regeneration in the defective area of the PDB group. Through extensive experimentation, we evaluated the scaffold’s effectiveness in augmenting osteoblast proliferation and differentiation. This study emphasized the potential of piezoelectric PVDF-b-PTFE nanofibrous membranes with controlled BMP-2 release as a promising approach for bone tissue engineering, providing a viable solution for addressing bone defects.

Biomimetic and spatiotemporally sequential hydrogel delivery system with self-healing and adhesion: Triple growth factor for bone defect repair

The healing of bone defects depends on early revascularization for delivery of oxygen, nutrients, growth factors, and stem cells to the injury region but also elimination of metabolic wastes. In this research, the combination of rapidly released VEGF and sustained release of PDGF-BB from the chitosan microspheres in low crosslinked outer layer promoted angiogenesis and vascularization, and sustained release of BMP-2 from the microspheres in high crosslinked inner layer provided structural support and facilitated osteogenic differentiation. In the skull defect model and the subcutaneous implantation model, the combination of triple growth factors significantly accelerated the regeneration of vascularized bone compared with stimulation of sole BMP-2 or only combination of VEGF and PDGF-BB. Notably, the antibacterial effect of QCS/PF hydrogel could effectively avoid the infection risk caused by surgical implantation of Gram-positive Staphylococcus aureus. In brief, the spatiotemporal specificity of the three growth factors was established in the “sandwich” structure QCS/PF (quaternized chitosan (QCS) and the aldehyde group of Pluronic®F127 (PF)) hydrogel delivery system, and the controlled and sequential release of the three growth factors within 28 days was achieved to better repair bone defects.

An injectable and thermosensitive hydrogel with nano-aided NIR-II phototherapeutic and chemical effects for periodontal antibacteria and bone regeneration

Periodontitis is a common public health problem worldwide and an inflammatory disease with irregular defect of alveolar bone caused by periodontal pathogens. Both antibacterial therapy and bone regeneration are of great importance in the treatment of periodontitis. In this study, injectable and thermosensitive hydrogels with 3D networks were used as carriers for controlled release of osteo-inductive agent (BMP-2) and Near Infrared Region-II (NIR-II) phototherapy agents (T8IC nano-particles). T8IC nano-particles were prepared by reprecipitation and acted as photosensitizer under 808 nm laser irradiation. Besides, we promoted photodynamic therapy (PDT) through adding H2O2 to facilitate the antibacterial effect instead of increasing the temperature of photothermal therapy (PTT). Hydrogel + T8IC + Laser + BMP-2 + H2O2 incorporated with mild PTT (45 °C), enhanced PDT and sustained release of BMP-2. It was present with excellent bactericidal effect, osteogenic induction and biosafety both in vitro and in vivo. Besides, immunohistochemistry staining and micro-CT analyses had confirmed that PTT and PDT could promote bone regeneration through alleviating inflammation state. Altogether, this novel approach with synergistic antibacterial effect, anti-inflammation and bone regeneration has a great potential for the treatment of periodontitis in the future.

A bioactive microparticle-loaded osteogenically enhanced bioprinted scaffold that permits sustained release of BMP-2.

Extrusion-based bioprinting technology is widely used for tissue regeneration and reconstruction. However, the method that uses only hydrogel as the bioink base material exhibits limited biofunctional properties and needs improvement to achieve the desired tissue regeneration. In this study, we present a three-dimensionally printed bioactive microparticle-loaded scaffold for use in bone regeneration applications. The unique structure of the microparticles provided sustained release of growth factor for > 4 weeks without the use of toxic or harmful substances. Before and after printing, the optimal particle ratio in the bioink for cell viability demonstrated a survival rate of ≥ 85% over 7 days. Notably, osteogenic differentiation and mineralization-mediated by human periosteum-derived cells in scaffolds with bioactive microparticles-increased over a 2-week interval. Here, we present an alternative bioprinting strategy that uses the sustained release of bioactive microparticles to improve biofunctional properties in a manner that is acceptable for clinical bone regeneration applications.

The effect of pore size on cell behavior in mesoporous bioglass scaffolds for bone regeneration

The material properties of bone tissue repair scaffolds dictate the interaction between the material and tissues or cells in vivo. The interface between them at the nanoscale can regulate cell adhesion, migration, and differentiation behaviors, thereby reshaping osteogenic properties of the material in vivo. In this study, a series of 3 nm, 8 nm and 20 nm mesoporous bioactive glass scaffolds (MSX-3, MSX-8 and MSX-20) were prepared by employing sol-gel technique and active self-assembly, with mesoporous bioglass serving as matrix. We then further examined effects of different mesopore pore sizes on the material biology of scaffolds and effects of osteogenic properties both in vitro and in vivo. The prepared multi-level micro-nano-structured bone repair scaffolds all had ∼300 μm macropores and possessed good biocompatibility and in vitro mineralizing activity. In vitro mineralization experiments proved that a large amount of calcium and phosphate salts were deposited on the surfaces of MSX-3 and MSX-8 scaffolds, while MSX-20 had conspicuous adsorption/deposition effects on organic compounds such as proteins in solution. MSX-20 multi-level micro-nano-structured bone repair scaffold could significantly promote the cell adhesion of BMSCs, and the cell spreading area was significantly larger than that of MSX-3 and MSX-8 scaffolds. MSX-20 multi-level micro-nano structured bone repair scaffold had a good sustained release effect on BMP-2, which could promote the osteogenic differentiation effect of BMP-2 on BMSCs. In vivo animal experiments also showed that MSX-20 loaded with BMP-2 could significantly promote in vivo osteogenesis. These findings suggest that when the mesopore pore size is 20 nm, the scaffold can be more conductive to bone regeneration due to the enhanced adhesion of stem cells and more sustained release of BMP-2.

A sustained release of BMP2 in urine-derived stem cells enhances the osteogenic differentiation and the potential of bone regeneration

Cell-based tissue engineering is one of the optimistic approaches to replace current treatments for bone defects. Urine-derived stem cells (USCs) are obtained non-invasively and become one of the promising seed cells for bone regeneration. An injectable BMP2-releasing chitosan microspheres/type I collagen hydrogel (BMP2-CSM/Col I hydrogel) was fabricated. USCs proliferated in a time-dependent fashion, spread with good extension and interconnected with each other in different hydrogels both for 2D and 3D models. BMP2 was released in a sustained mode for more than 28 days. Sustained-released BMP2 increased the ALP activities and mineral depositions of USCs in 2D culture, and enhanced the expression of osteogenic genes and proteins in 3D culture. In vivo, the mixture of USCs and BMP2-CSM/Col I hydrogels effectively enhanced bone regeneration, and the ratio of new bone volume to total bone volume was 38% after 8 weeks of implantation. Our results suggested that BMP2-CSM/Col I hydrogels promoted osteogenic differentiation of USCs in 2D and 3D culture in vitro and USCs provided a promising cell source for bone tissue engineering in vivo. As such, USCs-seeded hydrogel scaffolds are regarded as an alternative approach in the repair of bone defects.

A Biomimetic Macroporous Hybrid Scaffold with Sustained Drug Delivery for Enhanced Bone Regeneration.

Bone regeneration is a highly complex physiological process regulated by several factors. In particular, bone-mimicking extracellular matrix and available osteogenic growth factors such as bone morphogenetic protein (BMP) have been regarded as key contributors for bone regeneration. In this study, we developed a biomimetic hybrid scaffold (CEGH) with sustained release of BMP-2 that would result in enhanced bone formation. This hybrid scaffold, composed of BMP-2-loaded cryoelectrospun poly(ε-caprolactone) (PCL) (CE) surrounded by a macroporous gelatin/heparin cryogel (GH), is designed to overcome the drawbacks of the relatively weak mechanical properties of cryogels and poor biocompatibility and hydrophobicity of electrospun PCL. The GH component of the hybrid scaffold provides a hydrophilic surface to improve the biological response of the cells, while the CE component increases the mechanical strength of the scaffold to provide enhanced mechanical support for the defect area and a stable environment for osteogenic differentiation. After analyzing characteristics of the hybrid scaffold such as hydrophilicity, pore difference, mechanical properties, and surface charge, we confirmed that the hybrid scaffold shows enhanced cell proliferation rate and apatite formation in simulated body fluid. Then, we evaluated drug release kinetics of CEGH and confirmed the sustained release of BMP-2. Finally, the enhanced osteogenic differentiation of CEGH with sustained release of BMP-2 was confirmed by Alizarin Red S staining and real-time PCR analysis.

PEI-modified diatomite/chitosan composites as bone tissue engineering scaffold for sustained release of BMP-2

The bone healing defects resulting from bone disease remain a significant clinical challenge. The bone tissue engineering scaffolds combined with osteoinductive compounds represent an effective approach to overcome this challenge. In this study, a novel chitosan-based scaffold was prepared by incorporating modified natural diatomite (DE) as filler and adsorption element. Specifically, modified-diatomite (MDE) was synthesized by grafting polyethyleneimine (PEI) on the surface of diatomite via hydroxyl groups. The physicochemical characteristics of MDE, including chemical composition, zeta potential, and adsorption behavior, were investigated successively. Further, the mechanical strength, drug release, cytotoxicity and osteogenic activity analyses were carried out for the scaffold material. The FTIR and zeta potential analyses exhibited that the amino groups (–NH2) were grafted on MDE, and the surface potential of diatomite altered from −24 mV to 55 mV. Subsequently, the protein adsorption capacity and cytocompatibility of MDE were observed to be improved as compared to DE. The compressive strength was observed to be enhanced due to the addition of MDE. Besides, the composite scaffold loaded with rhBMP-2 demonstrated a more positive impact on proliferation and osteogenic differentiation of the bone mesenchymal stem cells, thus, indicating an optimal bone regeneration capacity. The findings obtained in this study reveal that the MDE-rhBMP-2/CS composite scaffold can be potentially used to promote the bone tissue regeneration.

Individualized plasticity autograft mimic with efficient bioactivity inducing osteogenesis

Mineralized tissue regeneration is an important and challenging part of the field of tissue engineering and regeneration. At present, autograft harvest procedures may cause secondary trauma to patients, while bone scaffold materials lack osteogenic activity, resulting in a limited application. Loaded with osteogenic induction growth factor can improve the osteoinductive performance of bone graft, but the explosive release of growth factor may also cause side effects. In this study, we innovatively used platelet-rich fibrin (PRF)-modified bone scaffolds (Bio-Oss®) to replace autograft, and used cytokine (BMP-2) to enhance osteogenesis. Encouragingly, this mixture, which we named “Autograft Mimic (AGM)”, has multiple functions and advantages. (1) The fiber network provided by PRF binds the entire bone scaffold together, thereby shaping the bone grafts and maintaining the space of the defect area. (2) The sustained release of BMP-2 from bone graft promoted bone regeneration continuously. (3) AGM recruited bone marrow mesenchymal stem cells (BMSCs) and promote their proliferation, migration, and osteogenic differentiation. Thus, AGM developed in this study can improve osteogenesis, and provide new guidance for the development of clinical bone grafts.

Osteogenic Effect of a Biodegradable BMP-2 Hydrogel Injected into a Cannulated Mg Screw.

Cannulated screws, containing an internal hole for inserting a guide pin, are commonly used in the management of bone fractures. Cannulated Mg screws can be biodegraded easily because their increased surface area including that of the inner hole rapidly reacts with body fluids. To delay biodegradation of cannulated Mg screws and improve bone regeneration, we developed a specific type of screw by injecting it with gelatin hydrogels [10 wt % gelatin(gel) with 0.09 v/v % glutaraldehyde (cross-linker)] containing different concentrations (5, 10, or 25 μg/mL) of bone morphogenic proteins (BMPs). We analyzed the properties and biocompatibility of the screws with and without BMP-2 and found that the release rate of BMP-2 in the hydrogel changed proportionately with the degradation rate of the cross-linked hydrogel. Loading BMP-2 in the hydrogel resulted in sustained release of BMP-2 for 25 to 40 days or more. The degradation rate of BMP-2 hydrogels was inversely proportional to the concentration of BMP-2. The injection of the hydrogels in the cannulated screw delayed biodegradation inside of the screw by simulated body fluid. It also induced uniform corrosion and the precipitation of bioactive compounds onto the surface of the screw. In addition, osteoblast proliferation was very active near the BMP-2 hydrogels, depending on the BMP-2 concentration. The BMP-2 in the hydrogel improved cell differentiation. The cannulated screw injected with 10 μL/mL BMP-2 hydrogel prevented implant biodegradation and enhanced osteoconduction and osteointegration inside and outside the screw. In addition, the properties of BMP-2-loaded hydrogels can be changed by controlling the amount of the cross-linker and protein, which could be useful for tissue regeneration in other fields.

Sustained protein therapeutics enabled by self-healing nanocomposite hydrogels for non-invasive bone regeneration.

Bone tissue engineering based on stem cells, growth factors and bioactive scaffolds presents an appealing but challenging approach for rehabilitation of patients with bone defects. A versatile system with the capability for easy operation and precise protein delivery in specific locations is attractive for enhancing bone regeneration. Here, we develop a non-invasive delivery system based on injectable and self-healing nanocomposite hydrogels for sustained protein release, which has the potential to improve the current orthopedic strategy. Specifically, LAPONITE® (LAP) nanoplatelets are able to accelerate the gelation process through hydrogen bonds with polysaccharide matrices, endowing hydrogels with superior mechanical and rheological behaviors, along with better injectability and self-healing ability. Attractively, the strong static binding between LAP nanoplatelets and bone morphogenetic protein-2 (BMP-2) can form stable LAP@BMP-2 complexes. The results indicate that the complexes effectively preserve the intrinsic bioactivity of BMP-2 and prolong the release period for more than four weeks. Moreover, hydrogels incorporating with the LAP@BMP-2 complexes synergistically boost cell spreading, proliferation activity and osteogenesis, both in vitro and in vivo, compared with LAP or BMP-2 alone. Overall, this study proposes a valid platform for protein therapeutics and non-invasive bone repair.

Sustained BMP-2 release and platelet rich fibrin synergistically promote tendon-bone healing after anterior cruciate ligament reconstruction in rat.

Anterior cruciate ligament (ACL) injuries which cause knee disabilities remain a clinical challenge due to the compromised tendon-bone repair. Multiple strategies have been proposed to treat the tendon-bone injuries, and the combination of these therapies hold great potential to achieve synergistic effects. We built PLGA-BMP-2 (bone morphogenetic protein 2) system and confirmed the sustained release of BMP-2 both in vitro and in vivo. We then applied different therapies to treat rat ACL reconstruction. We collected the tissue sample and analyzed the BMP-2 concentration both in serum and in injured sites. We tested the mRNA expression of genes that were related to inflammation, tissue repair and bone formation in damaged tissues. We also analyzed the protein levels of some genes associated with tendon formation and check the function of newly generated bone through biomechanical test. We found that, compared to monotherapies, simultaneous utilization of sustained BMP-2 release and platelet-rich fibrin (PRF) after anterior cruciate ligament reconstruction showed better therapeutic effects on tendon-bone healing in rat. This combined therapy efficiently enhanced the levels of growth factors that favor the angiogenesis and relieved the inflammatory responses in the injured sites. Of note, the combined therapy efficiently promoted the signals associated with bone formation and tendon regeneration. We demonstrated that the combined therapy with BMP-2 and PRF achieves synergistic effects on tendon-bone healing and holds great potential for the treatment of ACL reconstruction.

Controlled release of BMP-2 from titanium with electrodeposition modification enhancing critical size bone formation.

In this study, a porous Ti-alloy based implant with an interconnected channel structure (MAO-CaP-BMP2) is fabricated using a method combining 3D printing, microarc oxidation (MAO) treatment, and co-precipitation of Ca,P layer with BMP-2 technique. The macroporous structure with pore size of 600 μm made by 3D printing not only enhances the ingrowth of cells but also allows the formation of blood vessels inside the implant. As a result, the new bond formation is promoted. In addition, the microporous dioxide layer formed on the implant surface by MAO provides the sites for co-precipitation of Ca,P layer with BMP-2. The microstructure allows the prolonged release of BMP-2. Our results show that a sustained release of BMP-2 over 35 days is achieved for MAO-CaP-BMP2 group longer than Ti without MAO modification group and without Ca,P electrochemical deposition group. The slow release of BMP-2 at the bone/implant interface for a long period of time leads to enhancement of the osseointegration between the implant and surrounding bones. This result indicates that MAO-CaP-BMP2 is a good candidate of growth factor carrier. Successful regeneration of bone requires the concomitant processes of osteogenesis and neovascularization. MAO-CaP-BMP2 modified Ti-alloy implant is both osteoinductive and osteoconductive which can create better osteogenesis and angiogenesis. As a result, it can enhance bone formation.

Novel Surface Coatings Using Oxidized Glycosaminoglycans as Delivery Systems of Bone Morphogenetic Protein 2 (BMP-2) for Bone Regeneration.

Tissue engineering of bone requires the delivery of growth factors in a localized, sustained manner. Here, chitosan is used as polycation, while heparin and chondroitin sulfate are employed either as native or oxidized polyanions for formation of multilayers by layer-by-layer technique. The use of oxidized heparin and oxidized chondroitin sulfate permits additional stabilization by cross-linking through imine bond formation between amino groups of polycations and aldehydes of oxidized glycosaminoglycans (oGAGs). Since these multilayers are highly hydrophilic, adhesion of C2C12 myoblasts is improved either by the use of a specific 4+9pH regime with native glycosaminoglycans or a terminal collagen I layer in case of oGAGs. Adhesion and proliferation studies with C2C12 myoblasts, seeded either on bone morphogenetic protein (BMP-2) loaded or non-loaded multilayers, show that intrinsic cross-linking in oGAG-based multilayers supports cell adhesion, spreading, proliferation, and subsequent cell differentiation into osteoblasts. This is related to higher thickness and roughness of multilayers made of oGAGs compared to their native counterparts studied by ellipsometry and atomic force microscopy. Taken together, oGAG multilayer systems provide stable surface coatings and are useful as biocompatible reservoirs for sustained release of BMP-2, paving the way for coating implants and scaffolds for repair and regeneration of bone.

Chitosan/biphasic calcium phosphate scaffolds functionalized with BMP-2-encapsulated nanoparticles and RGD for bone regeneration.

Advancements in bone tissue engineering require the improvement of tissue scaffolds, which should not only exhibit suitable mechanical properties and highly porous structures, but also effectively carry signaling molecules that can mediate bone formation and tissue regeneration. In the present study, we established chitosan/biphasic calcium phosphate (CS/BCP) scaffolds functionalized with Arg-Gly-Asp (RGD) and BMP-2-loaded nanoparticles. The resulting scaffolds were highly similar to natural bone extracellular matrix (ECM) in terms of composition and structural properties. First, we synthesized CS/BCP composite bionic scaffolds via the freeze-drying method. Then, RGD peptides were covalently conjugated onto the scaffolds via the EDC/NHS method. The BMP-2-encapsulated BSA nanoparticles were prepared via a desolvation method and then coated with CS and oxidized alginate to achieve sustained release of BMP-2. In vitro cell culture and in vivo implantation tests confirmed that RGD and BMP-2 synergistically enhanced cell attachment and spreading by providing integrin binding surface and facilitating osteogenic differentiation. In summary, the bioceramic/biopolymer scaffolds functionalized with signaling biomolecules successfully provided a favorable microenvironment for bone formation and thus serve as potential candidates for use in bone tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2613-2624, 2018.

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.

Rapid initiation of guided bone regeneration driven by spatiotemporal delivery of IL-8 and BMP-2 from hierarchical MBG-based scaffold

Initiation of endogenous repair mechanisms, including key steps of stem cell recruitment and cartilage intermediate formation in endochondral ossification, is vital to regeneration of large bone defects. To biomimetically promote a rapid initiation and ensuing osteogenic stimulation, exogenous chemokine IL-8 and growth factor BMP-2 were orchestrated in a mesoporous bioactive glass (MBG)-based spatiotemporal delivery system, to achieve a rapid release of IL-8 followed by a long-term sustained release of BMP-2. The synergistic effect of IL-8 and BMP-2 on initiation stage of bone healing and underlying mechanism were thoroughly investigated in vitro and in vivo. Intriguingly, apart from its superiority in stem cell recruitment to BMP-2, IL-8 not only endowed a histological “prep-state” of endochondral ossification by up-regulating chondrogenic genes and inducing the formation of extensive cartilage tissues, facilitating rapid bone transformation by BMP-2, but also triggered a cellular “prep-state” with high expression of BMP receptors, enhancing the osteoinductivity of BMP-2. With the spatiotemporal delivery system, orchestrated signal stimuli of IL-8 and BMP-2 induced a rapid initiation including efficient stem cell recruitment and a “chondrogenic/osteogenic balance” at the first stage of endochondral ossification, and the scaffold facilitated sufficient osteoconductivity, together resulting in early extensive bone mineralization and an advanced regeneration throughout the repair of large bone defect. We believe this new idea could provide insights toward designing bone-repairing biomaterials with higher regenerative efficiency.

Abstract P29: Self Contained Bioreactor For Bone Regeneration

PURPOSE: The current gold standard for the repair of segmental bone defects is autologous bone grafts or flaps, however these are limited by donor site morbidity, limited graft tissue availability, and potential for complications at the harvest site. ASCs (adipose derived stem cells) have potential to aid bone regeneration due to their ability to differentiate into osteoblasts. BMP-2 (bone morphogenetic protein) has been shown to induce osteogenic differentiation of ASCs and enhance bone growth. We present a bioreactor for bone regeneration consisting of three main components: (i) a biodegradable PLLA scaffold seeded with ASCs, (ii) a drug reservoir made of PLLA and (iii) a controlled drug diffusion system for sustained release of BMP-2 to repair segmental bone defects. The drug reservoir is attached to the porous polymer scaffold seeded with ASCs and is capable of delivering growth factors for prolonged periods. METHODS: The components for the drug delivering setup were made from poly-lactic-acid (Purac Biomaterials) using solvent casting method. The device consists of two concentric tubes and a reservoir in between the tubes that stores BMP-2. Six 180 μm diffusion holes were drilled into the inner tube by pulsing a laser cutter. In vitro release tests were performed over a 30-day period. The concentration of BMP-2 released from devices was determined with an enzyme linked immunosorbent assay (R&D systems). The PLLA scaffolds were manufactured by a salt leaching method. ASC seeded scaffolds were inserted in the inner tube. We first evaluated the dosage(s) of BMP-2 that allows for the differentiation of ASCs into osteogenic cells using RT-PCR. ASC seeded devices were then loaded with BMP-2 and in vitro osteogenic differentiation of the ASCs was evaluated by direct delivery of BMP-2 from the drug reservoir to the seeded scaffold. RESULTS: The RT-PCR data showed increased expression of osteogenic markers such as ALK and Runx-2 in ASCs treated with 100ng/ml BMP-2 compared to no BMP-2 (p<0.05). Alizarin Red staining results showed that we were able to achieve osteogenic differentiation of ASCs with 100ng/ml BMP-2/day and the percentage of mineralization was greater in the PLLA scaffold compared to ‘no scaffold’ (p<0.05). The results for drug release kinetics showed that the drug delivery setup test was effective in achieving controlled local release of BMP-2 for 30 days. The results of the sealed (no hole) devices (n=2) validated sealing techniques for the drug-release reservoirs. The diffusion tests (n=6) indicated that 6x180 μm holes allowed for a sustainable and controlled BMP-2 release in the range of 50–100 ng/ml/day for 30 days. CONCLUSION: The bone bioreactor was able to release BMP-2 for 30 days in a controlled manner. The ASC seeded PLLA scaffold exposed to 50-100ng/m/day of BMP-2 produced significantly higher mineralization as compared to no scaffold. Further in vivo testing will be done to demonstrate that directly delivering BMP-2 from our reservoir to the ASC seeded scaffold enhances bone regeneration.

Is Heparin Effective for the Controlled Delivery of High-Dose Bone Morphogenetic Protein-2?

Sustained release of bone morphogenetic protein (BMP)-2 by heparin-contained biomaterials is advantageous for bone tissue regeneration using low-dose BMP-2. However, its effect with high-dose BMP-2 is still unclear and should be clarified considering the clinical use of a high dose of BMP-2 in spine and oral surgery. This study aimed to evaluate the efficacy of a heparin-conjugated collagen sponge (HCS) with high-dose BMP-2 delivery by investigating in vivo initial osteogenic regulation and bone healing over 12 weeks in comparison with that of an absorbable collagen sponge (ACS). The in vitro BMP-2 release profile in the HCS exhibited a lower burst followed by a sustained release of BMP-2, whereas that of the ACS showed an initial burst phase only. As a result of a lower burst, the HCS-BMP group showed higher expression of bone-forming/resorbing markers and enhanced activation of osteoclasts than the ACS-BMP group within the scaffold of defect after 7 days, which is presumed to be because of retention of relatively higher amounts of BMP-2. However, the surrounding calvariae were less resorbed in the HCS-BMP group, compared with the aggressive resorptive response in the ACS-BMP group. Microcomputed tomography and histology revealed that HCS-BMP guided more effective bone regeneration of central defect over time inducing minor ossification at the defect exterior, whereas ACS-BMP exhibited excessive ossification at the defect exterior. These results showed that HCS-mediated BMP-2 delivery at a high dose has advantages over ACS, including less early resorption of surrounding bone tissue and higher efficacy in compact bone regeneration over a longer period, highlighting a clinical feasibility of this technology.