Osteoinductive Growth Factors Research Articles

Effects of rhBMP-2 with various carriers on maxillofacial bone regeneration through computed tomography evaluation

BackgroundrhBMP-2 is regarded as the most potent osteoinductive growth factor, and it has been used in the oral cavity with different carriers. The purpose of this study is to evaluate the bone-regenerative effect of rhBMP-2 delivered with different carrier systems through three-dimensional cone beam computed tomography analysis.MethodA total of 112 patients underwent oral surgery with rhBMP-2 application (Group 1, n = 53) or without rhBMP-2 application (Group 2, n = 59). Group 1 was divided into 3 groups according to carriers, rhBMP-2 with allograft (Group 1–1, n = 34), rhBMP-2 with xenograft (Group 1–2, n = 5), and rhBMP-2 with absorbable collagen sponge (Group 1–3, n = 14). Cone beam computed tomography scans were taken before surgery (T0) 6 months after surgery (T1). The volume of defects was measured through the three-dimensional image analysis tool.ResultsThe average bone regeneration rate of Group 1 was significantly greater than that of Group 2. Within Group 1, the group that used allograft as a carrier (Group 1–1) showed significantly higher bone regeneration rates than the group that used absorbable collagen sponge as a carrier (Group 1–3).ConclusionThe use of rhBMP-2 after oral surgery results in a superior bone regeneration rate compared to not using rhBMP-2, and its efficacy depends on the carriers it is used with. Allograft affects bone regeneration more than absorbable collagen sponge when it is carried with rhBMP-2. Therefore, the appropriate use of rhBMP-2 with suitable bone grafting materials is useful for promoting postoperative bone regeneration in oral surgery.

A supramolecular polymer-collagen microparticle slurry for bone regeneration with minimal growth factor

Recombinant bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive growth factor that can promote bone regeneration for challenging skeletal repair and even for ectopic bone formation in spinal fusion procedures. However, serious clinical side effects related to supraphysiological dosing highlight the need for advances in novel biomaterials that can significantly reduce the amount of this biologic. Novel biomaterials could not only reduce clinical side effects but also expand the indications for use of BMP-2, while at the same time lowering the cost of such procedures. To achieve this objective, we have developed a slurry containing a known supramolecular polymer that potentiates BMP-2 signaling and porous collagen microparticles. This slurry exhibits a paste-like consistency that stiffens into an elastic gel upon implantation making it ideal for minimally invasive procedures. We carried out in vivo evaluation of the novel biomaterial in the rabbit posterolateral spine fusion model, and discovered efficacy at unprecedented ultra-low BMP-2 doses (5 μg/implant). This dose reduces the growth factor requirement by more than 100-fold relative to current clinical products. This observation is significant given that spinal fusion involves ectopic bone formation and the rabbit model is known to be predictive of human efficacy. We expect the novel biomaterial can expand BMP-2 indications for difficult cases requiring large volumes of bone formation or involving patients with underlying conditions that compromise bone regeneration.

DPSC-Derived Extracellular Vesicles Promote Rat Jawbone Regeneration

Repair and functional reconstruction of large jawbone defects remain one of the challenges in the field of head and neck surgery. The recent progress in tissue engineering technologies and stem cell biology has significantly promoted the development of regenerative reconstruction of jawbone defects. The multiple trophic activities of extracellular vesicles (EVs) produced by mesenchymal stem cells (MSCs) may play a critical role in their therapeutic effects. Accumulating evidence has shown the promise of dental pulp stem cells (DPSCs) in bone regeneration, but less is known about the regenerative effects of DPSC-EVs on jawbone defects. The purpose of this study is to explore the osteogenic effects of DPSC-EVs on jawbone marrow–derived MSCs (JB-MSCs) in vitro and their osteoinductive effects in a mandibular bone defect model in rats. Our results showed that JB-MSCs could efficiently uptake DPSC-EVs, which in turn significantly promoted the expression of osteogenic genes, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN), as well as the osteogenic differentiation capability of JB-MSCs. Meanwhile, we found that the pro-osteogenic effect in vitro induced by DPSC-EVs was comparable to that induced by BMP-2 (bone morphogenetic protein 2), currently the only Food and Drug Administration–approved osteoinductive growth factor. In vivo, animals that were locally treated with DPSC-EVs laden with a commercially available collagen membrane exhibited a relatively fast wound closure and increased new bone density at the mandible defects. Our results provide evidence for the osteogenic and osteoinductive effects of DPSC-EVs on jawbone regeneration. Due to the accessibility, rapid proliferation, and osteogenic propensity of DPSCs, DPSC-EVs may represent a safe cell-free therapeutic approach for craniofacial bone regeneration.

Arthrozheal®, a Bioactive Fibrin Scaffold for Joint Cartilage, Tendon and Soft Tissue Lesions. Latest Results and Application Perspectives.

Treatment of articular cartilage, tendon and soft tissue damage remains a challenge for the practicing orthopaedic surgeon. Due to the multifactorial aetiology of these lesions, there is a narrow therapeutic window within which they can be treated successfully, thus preventing progression to other musculoskeletal tissues. Recently, a new material that combines platelet-rich fibrin with collagen and is applied as a gel scaffold (ArthroZheal®, Vivostat A/S, Allerød, Denmark) has been shown to provide unique results in these patients. We arthroscopically treated 210 patients (114 knees, 32 hips, 52 shoulders, 12 ankle joints) with ArthroZheal®. The basic idea was to adjust treatment to the individual patient and to repair related and/or contributing problems before or along with treatment of chondral/tendon/ligament injuries. Arthroscopy was our preferred surgical method; the goal was to restore and preserve function, alleviate pain and minimise progression to osteoarthritis. We excluded cases of inflammatory arthropathy, unstable or malaligned joint, "kissing lesions" (bipolar), infection, obesity, massive rotator cuff rupture and multiligament instability. Our results were more than promising. We observed improved mobility in 93%, reduced pain in 95% at 3 months and further improvement at 6 months, with near-normal ROM (97% ) and pain-free status (98%). The MRI at 12 months post application showed cartilage restoration/reformation in 94% of patients, improved cartilage quality (84% )-by 2nd-look arthroscopic confirmationand normal tendon or ligament reconstruction (without stitching of the affected area)(95%). We were concerned about bone marrow oedema and rehab compliance among elderly patients. For successful regeneration of tissue lesions and osteochondral defects, natural gel bioscaffolds, combined with platelet rich fibrin (PRF) with chondroinductive and osteoinductive growth factor stimulators (ArthroZheal®) are required. There is no "gold standard" in the treatment of cartilage defect/tissue lesions or preferred treatment option. Many algorithms are used, which mostly rely on the surface area of the defect/site of lesion and on surgeon experience. An important issue is that rehabilitation depends on the treatment mode used and on the defect/lesion characteristics (classification and qualification). While a return to functional work and sports is possible with all procedures, different lengths of time are needed.

Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cell sheets co-expressing BMP2 and VEGF

Over-dependence on existing synthetic scaffolds and insufficient osteoinductive and vasculogenic growth factors have limited the development of bone regeneration. The study aimed to assess the feasibility of using marrow-derived mesenchymal stem cells (BMSCs) cell sheets co-expressing bone morphogenetic proteins 2 (BMP2) and vascular endothelial growth factor (VEGF) for repairing critical-sized calvarial defects. BMSCs cell sheets were genetically engineered to express BMP2/VEGF alone or together. Alterations in osteogenic markers were examined by quantitative real-time PCR (qRT-PCR) and western blotting. A critical-sized calvarial bone defect model was used to investigate the osteogenesis effects of BMP2/VEGF cell sheets alone or in combination. The efficacy was assessed with micro-computed tomography (micro-CT) and histology. Invitro, the expression of BMP2 and VEGF through lentiviral transduction was confirmed by qRT-PCR and western blotting against BMP2 and VEGF. Lentiviral delivery of BMP2 and VEGF resulted in the upregulation of osteogenic markers. Invivo, in a critical-sized calvarial bone defect model, 3D-reconstructed micro-CT images revealed that treatment of the calvarial defects with the BMP2/VEGF cell sheet resulted in significantly greater amounts of newly formed bone at 8 weeks after surgery than treatment with cell sheets with single gene transduction or vehicle controls. The results were confirmed by histological assessment by H&E staining and Masson staining. This study demonstrates that BMP2/VEGF co-expressing BMSCs sheets promote bone regeneration in critical-sized calvarial bone defects. The BMP2/VEGF cell sheets provide a functional bioactive scaffold for critical-size bone reconstruction.

Impact of oxygen-calcium-generating and bone morphogenetic protein-2 nanoparticles on survival and differentiation of bone marrow-derived mesenchymal stem cells in the 3D bio-printed scaffold

Although stem cell therapy is a major area of interest in tissue engineering, providing proper oxygen tension, good viability, and cell differentiation remain challenges in tissue-engineered scaffolds. In this study, an osteogenic scaffold was fabricated using the 3D bio-printing technique. The bio-ink contained alginate hydrogel, encapsulated human bone marrow-derived mesenchymal stem cells (hBM-MSCs), calcium peroxide nanoparticles (CPO NPs) as an oxygen generating biomaterial, and bone morphogenic protein-2 nanoparticles (BMP2 NPs) as an osteoinductive growth factor. CPO NPs were synthesized with the hydrolysis–precipitation method, and their concentrations in the bio-ink were optimized. Scaffolds containing CPO 3% (w/w) were preferred, because they generated sufficient oxygen gas for 20 days, increased mechanical strength after 20 days, and had sufficient stability. The CPO NPs effect on the viability of embedded hBM-MSCs under hypoxic conditions was analyzed. Live/Dead staining results represented a 22% improvement in CPO 3% scaffold viability on day 7. Therefore, CPO NPs constituted a promising survival factor. BMP2 NPs were prepared with the double emulsification technique. The incorporation of both BMP2 and CPO NPs resulted in the upregulation of Runt-related transcription factor 2, Collagen type I alpha 1, and the osteocalcin genes compared to internal references in osteogenic media. Overall, the proposed 3D bio-printed osteogenic scaffold in this study has moved scientific research one step forward toward successful stem cell therapy and helped improve host tissue healing by biological activity enhancement, especially for low oxygen pressure tissues.

Bone Healing Materials in the Treatment of Recalcitrant Nonunions and Bone Defects.

The usual treatment for bone defects and recalcitrant nonunions is an autogenous bone graft. However, due to the limitations in obtaining autogenous bone grafts and the morbidity associated with their procurement, various bone healing materials have been developed in recent years. The three main treatment strategies for bone defects and recalcitrant nonunions are synthetic bone graft substitutes (BGS), BGS combined with bioactive molecules, and BGS and stem cells (cell-based constructs). Regarding BGS, numerous biomaterials have been developed to prepare bone tissue engineering scaffolds, including biometals (titanium, iron, magnesium, zinc), bioceramics (hydroxyapatite (HA)), tricalcium phosphate (TCP), biopolymers (collagen, polylactic acid (PLA), polycaprolactone (PCL)), and biocomposites (HA/MONs@miR-34a composite coating, Bioglass (BG)-based ABVF-BG (antibiotic-releasing bone void filling) putty). Bone tissue engineering scaffolds are temporary implants that promote tissue ingrowth and new bone regeneration. They have been developed to improve bone healing through appropriate designs in terms of geometric, mechanical, and biological performance. Concerning BGS combined with bioactive molecules, one of the most potent osteoinductive growth factors is bone morphogenetic proteins (BMPs). In recent years, several natural (collagen, fibrin, chitosan, hyaluronic acid, gelatin, and alginate) and synthetic polymers (polylactic acid, polyglycolic acid, polylactic-coglycolide, poly(e-caprolactone) (PCL), poly-p-dioxanone, and copolymers consisting of glycolide/trimethylene carbonate) have been investigated as potential support materials for bone tissue engineering. Regarding BGS and stem cells (cell-based constructs), the main strategies are bone marrow stromal cells, adipose-derived mesenchymal cells, periosteum-derived stem cells, and 3D bioprinting of hydrogels and cells or bioactive molecules. Currently, significant research is being performed on the biological treatment of recalcitrant nonunions and bone defects, although its use is still far from being generalized. Further research is needed to investigate the efficacy of biological treatments to solve recalcitrant nonunions and bone defects.

Microgels based on Infernan, a glycosaminoglycan-mimetic bacterial exopolysaccharide, as BMP-2 delivery systems

Bone Morphogenetic Protein (BMP-2) is an osteoinductive growth factor clinically used for bone regeneration. Tuneable sustained strategies for BMP-2 delivery are intensely developed to avoid severe complications related to supraphysiological doses applied. To address this issue, we investigated the ability of the bacterial exopolysaccharide (EPS) called Infernan produced by the deep-sea hydrothermal vent bacterium Alteromonas infernus, exhibiting both glycosaminoglycan-mimetic and physical gelling properties, to efficiently bind and release the bioactive BMP-2. Two delivery systems were designed based on BMP-2 retention in either single or complex EPS-based microgels, both manufactured using a microfluidic approach. BMP-2 release kinetics were highly influenced by the ionic strength, affecting both microgel stability and growth factor/EPS binding, appearing essential for BMP-2 bioactivity. The osteogenic activity of human bone-marrow derived mesenchymal stem cells studied in vitro emphasized that Infernan microgels constitute a promising platform for BMP-2 delivery for further in vivo bone repair.

Engineering immunomodulatory and osteoinductive implant surfaces via mussel adhesion-mediated ion coordination and molecular clicking

Immune response and new tissue formation are important aspects of tissue repair. However, only a single aspect is generally considered in previous biomedical interventions, and the synergistic effect is unclear. Here, a dual-effect coating with immobilized immunomodulatory metal ions (e.g., Zn2+) and osteoinductive growth factors (e.g., BMP-2 peptide) is designed via mussel adhesion-mediated ion coordination and molecular clicking strategy. Compared to the bare TiO2 group, Zn2+ can increase M2 macrophage recruitment by up to 92.5% in vivo and upregulate the expression of M2 cytokine IL-10 by 84.5%; while the dual-effect of Zn2+ and BMP-2 peptide can increase M2 macrophages recruitment by up to 124.7% in vivo and upregulate the expression of M2 cytokine IL-10 by 171%. These benefits eventually significantly enhance bone-implant mechanical fixation (203.3 N) and new bone ingrowth (82.1%) compared to the bare TiO2 (98.6 N and 45.1%, respectively). Taken together, the dual-effect coating can be utilized to synergistically modulate the osteoimmune microenvironment at the bone-implant interface, enhancing bone regeneration for successful implantation.

Anterior Cervical Discectomy and Fusion Using Escherichia coli-Derived Recombinant Human Bone Morphogenetic Protein-2: A Pilot Study.

Recombinant human bone morphogenetic protein-2 (BMP-2) is an osteoinductive growth factor widely used in orthopedic surgery; it is also known to be associated with postoperative airway compromise or dysphagia when applied to anterior cervical discectomy and fusion (ACDF). However, there have been no reports on ACDF using Escherichia coli-derived BMP-2 (E.BMP-2) with hydroxyapatite (HA). This pilot study aimed to investigate the potential efficacy and safety of E.BMP-2 using HA as a carrier in ACDF prior to designing a larger-scale prospective study. Patients eligible for inclusion were those who underwent ACDF using 0.3 mg of E.BMP-2 with HA per segment for degenerative cervical disc disease between August 2019 and July 2020 and had at least 1 year of follow-up. Fusion rates were analyzed using computed tomography or flexion-extension radiographs. Visual analog scales for neck pain and arm pain and neck disability index were measured preoperatively and the final follow-up. In cases of cervical spondylotic myelopathy, modified Japanese Orthopaedic Association scores were also evaluated. Postoperative complications such as airway compromise, dysphagia, wound infection, neurologic deficit, hoarseness, heterotopic ossification, seroma, and malignancy were investigated. A total of 11 patients and 21 segments were analyzed. All clinical outcomes significantly improved at the final follow-up compared with the preoperative indices (p < 0.05). Only 1 case of dysphagia and no cases of airway compromise, wound infection, neurologic deficit, hoarseness, heterotopic ossification, seroma, or malignancy were observed during the follow-up period. Of the 21 segments, 15 segments showed solid fusion at 3 months after surgery, 4 segments at 6 months, and 1 segment at 12 months. Only 1 segment showed pseudoarthrosis, resulting in a fusion rate of 95.2%. The outcomes of ACDF could be enhanced using 0.3 mg of E.BMP-2 with HA per segment. Based on this study, larger-scale prospective studies can be conducted to evaluate the efficacy and safety of E.BMP-2 in ACDF.

Graphene oxide-functionalized nanocomposites promote osteogenesis of human mesenchymal stem cells via enhancement of BMP-SMAD1/5 signaling pathway

Biomaterials that can harness the intrinsic osteogenic potential of stem cells offer a promising strategy to accelerate bone regeneration and repair. Previously, we had used methacrylated gelatin (GelMA)-based scaffolds to achieve bone formation from human mesenchymal stem cells (hMSCs). In this study, we aimed to further enhance hMSC osteogenesis by incorporating graphene oxide (GO)-based nanosheets into GelMA. In vitro results showed high viability and metabolic activities in hMSCs encapsulated in the newly developed nanocomposites. Incorporation of GO markedly increased mineralization within hMSC-laden constructs, which was further increased by replacing GO with silica-coated graphene oxide (SiGO). Mechanistic analysis revealed that the nanosheet enhanced the production, retention, and biological activity of endogenous bone morphogenetic proteins (BMPs), resulting in robust osteogenesis in the absence of exogenous osteoinductive growth factors. Specifically, the osteoinductive effect of the nanosheets was abolished by inhibiting the BMP signaling pathway with LDN-193189 treatment. The bone formation potential of the technology was further tested in vivo using a mouse subcutaneous implantation model, where hMSCs-laden GO/GelMA and SiGO/GelMA samples resulted in bone volumes 108 and 385 times larger, respectively, than the GelMA control group. Taken together, these results demonstrate the biological activity and mechanism of action of GO-based nanosheets in augmenting the osteogenic capability of hMSCs, and highlights the potential of leveraging nanomaterials such as GO and SiGO for bone tissue engineering applications.

Investigation of a Prevascularized Bone Graft for Large Defects in the Ovine Tibia

In vivo bioreactors are a promising approach for engineering vascularized autologous bone grafts to repair large bone defects. In this pilot parametric study, we first developed a three-dimensional (3D) printed scaffold uniquely designed to accommodate inclusion of a vascular bundle and facilitate growth factor delivery for accelerated vascular invasion and ectopic bone formation. Second, we established a new sheep deep circumflex iliac artery (DCIA) model as an in vivo bioreactor for engineering a vascularized bone graft and evaluated the effect of implantation duration on ectopic bone formation. Third, after 8 weeks of implantation around the DCIA, we transplanted the prevascularized bone graft to a 5 cm segmental bone defect in the sheep tibia, using the custom 3D printed bone morphogenic protein 2 (BMP-2) loaded scaffold without prior in vivo bioreactor maturation as a control. Analysis by micro-computed tomography and histomorphometry found ectopic bone formation in BMP-2 loaded scaffolds implanted for 8 and 12 weeks in the iliac pouch, with greater bone formation occurring after 12 weeks. Grafts transplanted to the tibial defect supported bone growth, mainly on the periphery of the graft, but greater bone growth and less soft tissue invasion was observed in the avascular BMP-2 loaded scaffold implanted directly into the tibia without prior in vivo maturation. Histopathological evaluation noted considerably greater vascularity in the bone grafts that underwent in vivo maturation with an inserted vascular bundle compared with the avascular BMP-2 loaded graft. Our findings indicate that the use of an initial DCIA in vivo bioreactor maturation step is a promising approach to developing vascularized autologous bone grafts, although scaffolds with greater osteoinductivity should be further studied. Impact statement This translational pilot study aims at combining a tissue engineering scaffold strategy, in vivo prevascularization, and a modified transplantation technique to accelerate large segmental bone defect repair. First, we three-dimensional (3D) printed a 5 cm scaffold with a unique design to facilitate vascular bundle inclusion and osteoinductive growth factor delivery. Second, we established a new sheep deep circumflex iliac artery model as an in vivo bioreactor for prevascularizing the novel 3D printed osteoinductive scaffold. Subsequently, we transplanted the prevascularized bone graft to a clinically relevant 5 cm segmental bone defect in the sheep tibia for bone regeneration.

Application of Bone Morphogenetic Protein 7 Enhanced the Osteogenic Differentiation and Mineralization of Bone Marrow-Derived Stem Cells Cultured on Deproteinized Bovine Bone

The growth of bone morphogenetic protein 7 (BMP-7) has been applied for tissue regeneration due to its osteoinductive properties. The aim of this research is to analyze the enhancing effects of BMP-7 on the osteogenic differentiation and mineralization of human bone marrow-derived stem cells cultured on the bovine bone particle. After the stem cells were loaded onto the bone graft material, their morphology was observed on day 7. Viability assays based on the application of fluorescent stains were used for qualitative analyses. Alkaline phosphatase activity assays and Alizarin red staining were used for the assessment of osteogenic differentiation on days 7 and 14. Next-generation mRNA sequencing was applied to evaluate global gene expression. Gene ontology and pathway analysis was used to propose the underlying mechanism. Fibroblast-like morphology was attained with the stem cells. The cells were shown to be firmly attached to the bone particle. Most of the stem cells produced an intense green fluorescence. The relative cellular viability assay values for BMP-7 groups at 0, 10, and 100 ng/mL on day 7 were 0.295 ± 0.003, 0.250 ± 0.002, and 0.240 ± 0.003, respectively (p &lt; 0.05). Alkaline phosphatase activity was significantly higher in BMP-7 groups at concentration of 100 ng/mL compared to the control on days 7 and 14 (p &lt; 0.05). The results of the mineralization assay showed significantly higher values for BMP-7 groups at 100 ng/mL concentration when compared with the control (p &lt; 0.05). The expression of RUNX2 was increased with application of BMP-7 and mitogen-activated protein kinase pathway was associated with the target genes. Overall, this study shows that in vitro application of BMP-7 increases alkaline phosphorylase activity and mineralization of stem cells culture on deproteinized bovine bone mineral. The study suggests that combining stem cells with osteoinductive growth factors with scaffolds can have synergy effects on osteogenic differentiation.

Bone morphogenetic protein 2: heterologous expression and potential in bone regeneration

Currently, bone morphogenetic protein 2 (BMP-2) is one of the two osteoinductive growth factors used in medical devices to promote bone formation. Typically, this protein is bought from commercial houses at high rates and in small quantities that are not enough to cover clinical needs. Because of this, it has been proposed that research centers use their own heterologous expression systems to have a constant supply of BMP-2. The aim of this study was to standardize the heterologous expression of BMP-2 and evaluate its osteoinductive activity in vitro. Our procedure for expression and purification was based on recombinant DNA technology using the plasmid pET-28 and IPTG as inductor. After extracting the protein from inclusion bodies, folding it and modifying it via a redox system, we observed via electrophoresis a 26 kDa dimer. We evaluated its osteoinductive activity in myoblastic C2C12 by quantifying enzymatically the activity of alkaline phosphate (ALP) and staining mineralization nodules. ALP activity is proportional to BMP-2 concentration, increasing 90% at 3 µg/mL. These cells form calcium nodules, mineralizing 50% of the area.

High-modulus nanocomposite scaffold based on waterborne polyurethane grafted collagen polypeptide/hydroxyapatite for potential bone healing

A potential nanocomposite scaffold based on waterborne polyurethane grafted collagen polypeptide/hydroxyapatite (WPU-g-CP/HAp) was investigated in this research. First, a non-solvated WPU-g- CP copolymer emulsion was synthesized via water phase cross-linking copolymerization between waterborne polyurethane (WPU) prepolymer and KH550 modified collagen polypeptide (CP). Then, the WPU-g-CP/HAp was fabricated employing bionic precipitation method of HAp nanocrystals, followed by freeze-drying casting. TEM images of WPU-g-CP/HAp emulsion particles showed an assembly of polymer nano-microspheres with a HAp mineralized area. FT-IR and XRD results confirmed that the large quantities of HAP microcrystalline structures were formed in the WPU-g-CP/HAp nanocomposite. DMA analysis demonstrated that the WPU-g-CP/HAp had extremely high storage modulus (7000 MPa) and mechanical strength, which were very important for mechanical support as an ideal bone healing material. TG/DTG, SEM and EDS indicated that HAp has been uniformly dispersed into the nanocomposite in the form of crystallites, and showed good thermal stability and structural stability. Biodegradation test revealed that the degradation cycle of WPU-g-CP/HAp can meet the requirements of bone healing material. It could be expected that in the process of nanocomposite degradation the HAp will be continuously released into the extracellular environment to achieve bone healing via providing scaffolding for osteoconduction, proliferation, growth factors for osteoinduction, and new bone formation, which is extremely beneficial as a bone tissue healing scaffold.

The Effects of Stably Tethered BMP-2 on MC3T3-E1 Preosteoblasts Encapsulated in a PEG Hydrogel

Bone morphogenetic protein-2 (BMP-2) is a clinically used osteoinductive growth factor. With a short half-life and side effects, alternative delivery approaches are needed. This work examines thiolation of BMP-2 for chemical attachment to a poly(ethylene glycol) hydrogel using thiol-norbornene click chemistry. BMP-2 retained bioactivity post-thiolation and was successfully tethered into the hydrogel. To assess tethered BMP-2 on osteogenesis, MC3T3-E1 preosteoblasts were encapsulated in matrix metalloproteinase (MMP)-sensitive hydrogels containing RGD and either no BMP-2, soluble BMP-2 (5 nM), or tethered BMP-2 (40-200 nM) and cultured in a chemically defined medium containing dexamethasone for 7 days. The hydrogel culture supported MC3T3-E1 osteogenesis regardless of BMP-2 presentation, but tethered BMP-2 augmented the osteogenic response, leading to significant increases in osteomarkers, Bglap and Ibsp. The ratio, Ibsp-to-Dmp1, highlighted differences in the extent of differentiation, revealing that without BMP-2, MC3T3-E1 cells showed a higher expression of Dmp1 (low ratio), but an equivalent expression with tethered BMP-2 and more abundant bone sialoprotein. In addition, this work identified that dexamethasone contributed to Ibsp expression but not Bglap or Dmp1 and confirmed that tethered BMP-2 induced the BMP canonical signaling pathway. This work presents an effective method for the modification and incorporation of BMP-2 into hydrogels to enhance osteogenesis.

Prevascularized hydrogels with mature vascular networks promote the regeneration of critical-size calvarial bone defects in vivo.

Adequate vascularization of scaffolds is a prerequisite for successful repair and regeneration of lost and damaged tissues. It has been suggested that the maturity of engineered vascular capillaries, which is largely determined by the presence of functional perivascular mural cells (or pericytes), plays a vital role in maintaining vessel integrity during tissue repair and regeneration. Here, we investigated the role of pericyte-supported-engineered capillaries in regenerating bone in a critical-size rat calvarial defect model. Prior to implantation, human umbilical vein endothelial cells and human bone marrow stromal cells (hBMSCs) were cocultured in a collagen hydrogel to induce endothelial cell morphogenesis into microcapillaries and hBMSC differentiation into pericytes. Upon implantation into the calvarial bone defects (8mm), the prevascularized hydrogels showed better bone formation than either untreated controls or defects treated with autologous bone grafts (positive control). Bone formation parameters such as bone volume, coverage area, and vascularity were significantly better in the prevascularized hydrogel group than in the autologous bone group. Our results demonstrate that tissue constructs engineered with pericyte-supported vascular capillaries may approximate the regenerative capacity of autologous bone, despite the absence of osteoinductive or vasculogenic growth factors.

Microsphere embedded hydrogel construct - binary delivery of alendronate and BMP-2 for superior bone regeneration.

Biomimetic delivery of osteoinductive growth factors via an osteoconductive matrix is an interesting approach for stimulating bone regeneration. In this context, the bone extracellular matrix (ECM) has been explored as an optimal delivery system, since it releases growth factors in a spatiotemporal manner from the matrix. However, a bone ECM hydrogel alone is weak, unstable, and prone to microbial contamination and also has been reported to have significantly reduced bone morphogenic protein-2 (BMP-2) post decellularization. In the present work, a microsphere embedded osteoinductive decellularized bone ECM/oleoyl chitosan based hydrogel construct (BOC) was developed as a matrix allowing dual delivery of an anti-resorptive drug (alendronate, ALN, via the microspheres) and BMP-2 (via the hydrogel) for a focal tibial defect in a rabbit model. The synthesized gelatin microspheres (GMs) were spherical in shape with diameter ∼32 μm as assessed by SEM analysis. The BOC construct showed sustained release of ALN and BMP-2 under the studied conditions. Interestingly, amniotic membrane-derived stem cells (HAMSCs) cultivated on the hydrogel construct demonstrated excellent biocompatibility, cell viability, and active proliferation potential. Additionally, cell differentiation on the constructs showed an elevated expression of osteogenic genes in an RT-PCR study along with enhanced mineralized matrix deposition as demonstrated by alkaline phosphatase (ALP) assay and alizarin red assay. The hydrogel construct was witnessed to have improved neo-vascularization potential in a chick chorioalantoic membrane (CAM) assay. Also, histological and computed tomographic findings evidenced enhanced bone regeneration in the group treated with the BOC/ALN/BMP hydrogel construct in a rabbit tibial defect model. To conclude, the developed multifunctional hydrogel construct acts as an osteoinductive and osteoconductive platform facilitating controlled delivery of ALN and BMP-2, essential for stimulating bone tissue regeneration.

PEGylated Coating Affects DBM Osteoinductivity In Vivo by Changing Inflammatory Responses

PEGylation is a widely used modification in device coating or drug delivery by combing materials with poly(ethylene glycol) (PEG). In the present study, a well-established rat ectopic bone formation model was used to elucidate how PEGylated coating affects demineralized bone matrix (DBM) osteoinductivity in vivo by changing the inflammation events at the early phase of implantation. A range of cell-matrix interactions was characterized at the cellular and functional levels, including growth factor activity and kinetics, immune cell migration and activation, and bone formation in vivo. After 28 days, DBM's bone formation potential decreased in groups with increasing PEG concentration in the gelatin carrier. The increasing PEG concentration did not affect DBM's osteoinductive growth factor release or activity. However, increasing PEG cross-linking concentration resulted in decreased DBM-related early phase inflammatory reactions, reduced neutrophil infiltration, decreased coating material degradation, lowered the total number and active mast cells, and decreased CD80+ macrophage expression. Understanding and controlling cell-material responses may improve the design and development of functional medical devices.

Triple Growth Factor Delivery Promotes Functional Bone Regeneration Following Composite Musculoskeletal Trauma

Type III fractures typically involve segmental bone loss with extensive adjacent soft tissue injury to muscle and vasculature. Such severe composite injuries to bone and muscle often require multiple treatment procedures and are associated with significantly higher rates of complications, including non-union, infection, prolonged disability, and amputation. Successful bone healing depends on early re-vascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote re-vascularization following severe bone injuries, however results have been inconsistent. In this study, co-stimulation of microvascular fragment constructs with vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined delivery of VEGF and PDGF with a low dose of bone morphogenetic protein-2 (BMP-2) significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. This study demonstrates the potential for a combined osteoinductive and angiogenic growth factor delivery strategy to promote functional bone regeneration following severe composite musculoskeletal injury.