Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology.
By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.
With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.
Biomaterial scaffolds that deliver growth factors such as recombinant human bone morphogenetic proteins-2 (rhBMP-2) have improved clinical bone tissue engineering by enhancing bone tissue regeneration. This approach could be further improved if the controlled delivery of bioactive rhBMP-2 were sustained throughout the duration of osteogenesis from fibrous scaffolds that provide control over dose and bioactivity of rhBMP-2. In nature, heparan sulfate attached to core proteoglycans serves as the co-receptor that delivers growth factors to support tissue morphogenesis.
To mimic this behavior, we conjugated heparan sulfate decorated recombinant domain I of perlecan/HSPG2 onto an electrospun poly(ε-caprolactone) (PCL) scaffold, hypothesizing that the heparan sulfate chains will enhance rhBMP-2 loading onto the scaffold and preserve delivered rhBMP-2 bioactivity.
In this study, we demonstrated that covalently conjugated perlecan domain I increased loading capacity of rhBMP-2 onto PCL scaffolds when compared to control unconjugated scaffolds. Additionally, rhBMP-2 released from the modified scaffolds enhanced alkaline phosphatase activity in W20–17 mouse bone marrow stromal cells, indicating the preservation of rhBMP-2 bioactivity indicative of osteogenesis.
We conclude that this platform provides a sophisticated and efficient approach to deliver bioactive rhBMP-2 for bone tissue regeneration applications.
Electronic supplementary material
The online version of this article (doi:10.1186/s40634-016-0057-1) contains supplementary material, which is available to authorized users.
Heparan sulfate; Poly(ε-caprolactone); Bone morphogenetic protein; Alkaline phosphatase; Perlecan/HSPG2; Bone regeneration
The goal of this study was to use bioengineered injectable microgels to enhance the action of bone morphogenetic protein 2 (BMP2) and stimulate cartilage matrix repair in a reversible animal model of osteoarthritis (OA). A module of perlecan (PlnD1) bearing heparan sulfate (HS) chains was covalently immobilized to hyaluronic acid (HA) microgels for the controlled release of BMP2 in vivo. Articular cartilage damage was induced in mice using a reversible model of experimental OA and was treated by intra-articular injection of PlnD1-HA particles with BMP2 bound to HS. Control injections consisted of BMP2 free PlnD1-HA particles, HA particles, free BMP2 or saline. Knees dissected following these injections were analyzed using histological, immunostaining and gene expression approaches. Our results show that knees treated with PlnD1-HA/BMP2 had lesser OA-like damage compared to control knees. In addition, the PlnD1-HA/BMP2-treated knees had higher mRNA levels encoding for type II collagen, proteoglycans, and xylosyltransferase 1, a rate-limiting anabolic enzyme involved in the biosynthesis of glycosaminoglycan chains, relative to control knees (PlnD1-HA). This finding was paralleled by enhanced levels of aggrecan in the articular cartilage of PlnD1-HA/BMP2 treated knees. Additionally, decreases in the mRNA levels encoding for cartilage-degrading enzymes and type X collagen were seen relative to controls. In conclusion, PlnD1-HA microgels constitute a formulation improvement compared to HA for efficient in vivo delivery and stimulation of proteoglycan and cartilage matrix synthesis in mouse articular cartilage. Ultimately, PlnD1-HA/BMP2 may serve as an injectable therapeutic agent for slowing or inhibiting the onset of OA after knee injury.
Perlecan; Hyaluronic Acid; Heparan Sulfate; Osteoarthritis; Cartilage Repair; Bone Morphogenetic Protein
Strategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.
Canine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.
In vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.
An intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.
Autologous bone grafting remains the gold standard in the treatment of large bone defects but is limited by tissue availability and donor site morbidity. Recombinant human bone morphogenetic protein-2 (rhBMP-2), delivered with a collagen sponge, is clinically used to treat large bone defects and complications such as delayed healing or nonunion. For the same dose of rhBMP-2, we have shown that a hybrid nanofiber mesh-alginate (NMA-rhBMP-2) delivery system provides longer-term release and increases functional bone regeneration in critically sized rat femoral bone defects compared with a collagen sponge. However, no comparisons of healing efficiencies have been made thus far between this hybrid delivery system and the gold standard of using autograft.
We compared the efficacy of the NMA-rhBMP-2 hybrid delivery system to morselized autograft and hypothesized that the functional regeneration of large bone defects observed with sustained BMP delivery would be at least comparable to autograft treatment as measured by total bone volume and ex vivo mechanical properties.
Bilateral critically sized femoral bone defects in rats were treated with either live autograft or with the NMA-rhBMP-2 hybrid delivery system such that each animal received one treatment per leg. Healing was monitored by radiography and histology at 2, 4, 8, and 12 weeks. Defects were evaluated for bone formation by longitudinal micro-CT scans over 12 weeks (n = 14 per group). The bone volume, bone density, and the total new bone formed beyond 2 weeks within the defect were calculated from micro-CT reconstructions and values compared for the 2-, 4-, 8-, and 12-week scans within and across the two treatment groups. Two animals were used for bone labeling with subcutaneously injected dyes at 4, 8, and 12 weeks followed by histology at 12 weeks to identify incremental new bone formation. Functional recovery was measured by ex vivo biomechanical testing (n = 9 per group). Maximum torque and torsional stiffness calculated from torsion testing of the femurs at 12 weeks were compared between the two groups.
The NMA-rhBMP-2 hybrid delivery system resulted in greater bone formation and improved biomechanical properties compared with autograft at 12 weeks. Comparing new bone volume within each group, the NMA-rhBMP-2-treated group had higher volume (p < 0.001) at 12 weeks (72.59 ± 18.34 mm3) compared with 8 weeks (54.90 ± 16.14) and 4 weeks (14.22 ± 9.59). The new bone volume was also higher at 8 weeks compared with 4 weeks (p < 0.001). The autograft group showed higher (p < 0.05) new bone volume at 8 weeks (11.19 ± 8.59 mm3) and 12 weeks (14.64 ± 10.36) compared with 4 weeks (5.15 ± 4.90). Between groups, the NMA-rhBMP-2-treated group had higher (p < 0.001) new bone volume than the autograft group at both 8 and 12 weeks. Local mineralized matrix density in the NMA-rhBMP-2-treated group was lower than that of the autograft group at all time points (p < 0.001). Presence of nuclei within the lacunae of the autograft and early appositional bone formation seen in representative histology sections suggested that the bone grafts remained viable and were functionally engrafted within the defect. The bone label distribution from representative sections also revealed more diffuse mineralization in the defect in the NMA-rhBMP-2-treated group, whereas more localized distribution of new mineral was seen at the edges of the graft pieces in the autograft group. The NMA-rhBMP-2-treated group also revealed higher torsional stiffness (0.042 ± 0.019 versus 0.020 ± 0.022 N-m/°; p = 0.037) and higher maximum torque (0.270 ± 0.108 versus 0.125 ± 0.137 N-m; p = 0.024) compared with autograft.
The NMA-rhBMP-2 hybrid delivery system improved bone formation and restoration of biomechanical function of rat segmental bone defects compared with autograft treatment.
Delivery systems that allow prolonged availability of BMP may provide an effective clinical alternative to autograft treatment for repair of segmental bone defects. Future studies in a large animal model comparing mixed cortical-trabecular autograft and the NMA-rhBMP-2 hybrid delivery system are the next step toward clinical translation of this approach.
Commercially available recombinant human bone morphogenetic protein 2 (rhBMP2) has demonstrated efficacy in bone regeneration, but not without significant side effects. In this study, we utilize rhBMP2 encapsulated in PLGA microspheres (PLGA-rhBMP2) placed in a rabbit cranial defect model to test whether low-dose, sustained, delivery can effectively induce bone regeneration.
rhBMP2 was encapsulated in 15% poly (lactic-co-glycolic acid), using a double emulsion, solvent extraction/evaporation technique, and its release kinetics and bioactivity were tested. Two critical-size defects (10mm) were created in the calvarium of New Zealand White rabbits (5-7 mos of age, M/F) and filled with a collagen scaffold containing one of four groups: 1) no implant, 2) collagen scaffold only, 3) PLGA-rhBMP2(0.1ug/implant), or 4) free rhBMP2 (0.1ug/implant). After 6 weeks, the rabbits were sacrificed and defects were analyzed by μCT, histology, and finite element analysis.
RhBMP2 delivered via bioactive PLGA microspheres resulted in higher volumes and surface area coverage of new bone than an equal dose of free rhBMP2 by μCT and histology (p=0.025, 0.025). FEA indicated that the mechanical competence using the regional elastic modulus did not differ with rhBMP2 exposure (p=0.70). PLGA-rhBMP2 did not demonstrate heterotopic ossification, craniosynostosis, or seroma formation.
Sustained delivery via PLGA microspheres can significantly reduce the rhBMP2 dose required for de novo bone formation. Optimization of the delivery system may be a key to reduce the risk for recently reported rhBMP2 related adverse effects.
Level of Evidence
Injectable bone substitutes and techniques have been developed for use in minimally invasive procedures for bone augmentation.
: To develop a novel injectable thermo-sensitive alginate hydrogel (TSAH) as a scaffold to induce bone regeneration, using a minimally invasive tunnelling technique.
Material and Methods
: An injectable TSAH was prepared from a copolymer solution of 8.0 wt% Poly(N-isopropylacrylamide) (PNIPAAm) and 8.0 wt% AAlg-g-PNIPAAm. In vitro properties of the material, such as its microstructure and the sustained release of recombinant human bone morphogenetic protein-2 (rhBMP-2), were investigated. Then, with the subperiosteal tunnelling technique, this material, carrying rhBMP-2, was injected under the labial periosteum of the maxillary anterior alveolar ridge in a rabbit model. New bone formation was evaluated by means of X-ray, micro-computed tomography (micro-CT), fluorescence labelling, histological study, and immunohistochemistry study.
: The material exhibited good injectability and thermo-irreversible properties. SEM showed an interconnected porous microstructure of the TSAH. The result of ALP activity indicated sustained delivery of BMP-2 from the TSAH from days 3 to 15. In a rabbit model, both TSAH and TSAH/rhBMP-2 induced alveolar ridge augmentation. The percentage of mineralised tissue in the TSAH/rhBMP-2 group (41.6±3.79%) was significantly higher than in the TSAH group (31.3±7.21%; p<0.05). The density of the regenerating tissue was higher in the TSAH/rhBMP-2 group than in the other groups (TSAH group, positive control, blank control; p<0.05).
: The TSAH provided convenient handling properties for clinical application. To some extent, TSAH could induce ridge augmentation and mineral deposition, which can be enhanced when combined with rhBMP-2 for a minimally invasive tunnelling injection.
Alveolar ridge augmentation; Minimally invasive surgical procedures; Hydrogel; Tissue engineering
Perlecan, a heparan sulfate proteoglycan, is widely distributed in developing and adult tissues and plays multiple, important physiological roles. Studies with knockout mouse models indicate that expression of perlecan and heparan sulfate is critical for proper skeletal morphogenesis. Heparan sulfate chains bind and potentiate the activities of various growth factors such as fibroblast growth factor 2 (FGF-2). Previous studies indicate that important biological activities are associated with the heparan sulfate-bearing domain I of perlecan (PlnDI; French et al. J. Bone Miner. Res. 17, 48, 2002). In the present study, we have used recombinant, glycosaminoglycan-bearing PlnDI to reconstitute three-dimensional scaffolds of collagen I. Collagen I fibrils bound PlnDI much better than native collagen I monomers or heat-denatured collagen I preparations. Heparitinase digestion demonstrated that recombinant PlnDI was substituted with heparan sulfate and that these heparan sulfate chains were critically important not only for efficient integration of PlnDI into scaffolds, but also for FGF-2 binding and retention. PlnDI-containing collagen I scaffolds to which FGF-2 was bound sustained growth of both MG63, an osteoblastic cell line, and human bone marrow stromal cells (hBMSCs) significantly better than scaffolds lacking either PlnDI or FGF-2. Collectively, these studies demonstrate the utility of PlnDI in creating scaffolds that better mimic natural extracellular matrices and better support key biological activities.
A promising strategy to accelerate joint implant integration and reduce recovery time and failure rates is to deliver a combination of certain growth factors to the integration site. There is a need to control the quantity of growth factors delivered at different times during the healing process to maximize efficacy. Polyelectrolyte multilayer (PEM) films, built using the layer-by-layer (LbL) technique, are attractive for releasing controlled amounts of potent growth factors over a sustained period. Here, we present PEM films that sequester physiological amounts of osteogenic rhBMP-2 (recombinant human bone morphogenetic protein - 2) and angiogenic rhVEGF165 (recombinant human vascular endothelial growth factor) in different ratios in a degradable [poly(β-amino ester)/polyanion/growth factor/ polyanion] LbL tetralayer repeat architecture where the biologic load scaled linearly with the number of tetralayers. No burst release of either growth factor was observed as the films degraded. The release of rhBMP-2 was sustained over a period of 2 weeks, while rhVEGF165 eluted from the film over the first 8 days. Both growth factors retained their efficacy, as quantified with relevant in vitro assays. rhBMP-2 initiated a dose dependent differentiation cascade in MC3T3-E1S4 pre-osteoblasts while rhVEGF165 upregulated HUVEC proliferation, and accelerated closure of a scratch in HUVEC cell cultures in a dose dependent manner. In vivo, the mineral density of ectopic bone formed de novo by rhBMP-2/rhVEGF165 PEM films was approximately 33% higher than when only rhBMP-2 was introduced, with a higher trabecular thickness, which would indicate a decrease in the risk of osteoporotic fracture. Bone formed throughout the scaffold when both growth factors were released, which suggests more complete remodeling due to an increased local vascular network. This study demonstrates a promising approach to delivering precise doses of multiple growth factors for a variety of implant applications where control over spatial and temporal release profile of the biologic is desired.
Controlled drug release; BMP; VEGF; bone; hip replacement prosthesis; layer-by-layer; polyelectrolyte multilayer; dose response
It is advantageous to incorporate controlled growth factor delivery into tissue engineering strategies. The objective of this study was to develop a three-dimensional (3D) porous tissue engineering scaffold with the capability of controlled releasing recombinant human bone morphogenetic protein-7 (rhBMP-7) for enhancement of bone regeneration. RhBMP-7 was first encapsulated into poly(lactic-co-glycolic acid) (PLGA) nanospheres (NS) with an average diameter of 300 nm. Poly(L-lactic acid) (PLLA) scaffolds with interconnected macroporous and nano-fibrous architectures were prepared using a combined sugar sphere template leaching and phase separation technique. A post-seeding technique was then utilized to immobilize rhBMP-7 containing PLGA nanospheres onto prefabricated nano-fibrous PLLA scaffolds with well maintained 3D structures. In vitro release kinetics indicated that nanosphere immobilized scaffold (NS-scaffold) could release rhBMP-7 in a temporally controlled manner, depending on the chemical and degradation properties of the nanospheres which were immobilized onto the scaffold. In vivo, rhBMP-7 delivered from NS-scaffolds induced significant ectopic bone formation throughout the scaffold while passive adsorption of rhBMP-7 into the scaffold resulted in failure of bone induction due to either the loss of rhBMP-7 biological function or insufficient duration within the scaffold. We conclude that the interconnected macroporous architecture and the sustained, prolonged delivery of bioactive rhBMP-7 from NS immobilized nano-fibrous scaffolds actively induced new bone formation throughout the scaffold. The approach offers a new delivery method of BMPs and a novel scaffold design for bone regeneration.
C3H10T1/2 cells differentiate along a chondrogenic pathway when plated onto the extracellular matrix (ECM) protein perlecan (Pln). To identify the region(s) within the large Pln molecule that provides a differentiation signal, recombinant Pln-sequence-based polypeptides representing distinct structural domains were assayed for their ability to promote chondrogenesis in C3H10T1/2 cells. Five distinct domains, along with structural variations, were tested. The N-terminal domain I was tested in two forms (IA and IB) that contain only heparan sulfate (HS) chains or both HS and chondroitin sulfate (CS) chains, respectively. A mutant form of domain I lacking attachment sites for both HS and CS (Pln Imut) was tested also. Other constructs consecutively designated Pln domains II, III(A-C), IV(A,B), and V(A,B) were used to complete the structure-function analysis. Cells plated onto Pln IA or Pln IB but no other domain rapidly assembled into cellular aggregates of 40-120 μm on average. Aggregate formation was dependent on the presence of glycosaminoglycan (GAG) chains, because Pln I-based polypeptides lacking GAG chains either by enzymatic removal or mutation of HS/CS attachment sites were inactive. Aggregates formed on GAG-bearing Pln IA stained with Alcian Blue and were recognized by antibodies to collagen type II and aggrecan but were not recognized by an antibody to collagen type X, a marker of chondrocyte hypertrophy. Collectively, these studies indicate that the GAG-bearing domain I of Pln provides a sufficient signal to trigger C3H10T1/2 cells to enter a chondrogenic differentiation pathway. Thus, this matrix proteoglycan (PG) found at sites of cartilage formation in vivo is likely to enhance early stage differentiation induced by soluble chondrogenic factors.
perlecan; cartilage; chondrogenesis; proteoglycan
Extracellular matrix (ECM) molecules in cartilage, cooperate with growth factors to regulate chondrogenic differentiation and cartilage development. Domain I of perlecan (Pln) bears heparan sulfate chains that bind and release heparin binding growth factors (HBGFs). Our hypothesis was that Pln domain I (PlnDI) might be complexed with collagen II (P-C) fibrils to improve binding of bone morphogenetic protein-2 (BMP-2) and better support chondrogenesis and cartilage-like tissue formation in vitro. Our results showed that P-C fibrils bound more BMP-2 than collagen II fibrils alone, and better sustained BMP-2 release. Polylactic acid (PLA)-based scaffolds coated with P-C fibrils immobilized more BMP-2 than either PLA scaffolds or PLA scaffolds coated with collagen II fibrils alone. Multipotential mouse embryonic mesenchymal cells, C3H10T1/2, were cultured on two-dimensional P-C fibrils or three dimensional P-C/BMP-2-coasted (P-C-B) PLA scaffolds. Chondrogenic differentiation was indexed by glycosaminoglycan (GAG) production, and expression of the pro-chondrogenic transcription factor, Sox9, as well as cartilaginous ECM proteins, collagen II and aggrecan. Immunostaining for aggrecan, perlecan, tenascin and collagen X revealed that both C3H10T1/2 cells and primary mouse embryonic fibroblasts cultured on P-C-B fibrils showed the highest expression of chondrogenic markers among all treatment groups. Safranin O-Fast Green staining indicated that cartilage-like tissue was formed in the P-C-B scaffolds, while no obvious cartilage-like tissue formed in other scaffolds. We have concluded that P-C fibrils provide an improved biomimetic material for the binding and retention of BMP-2 and support chondrogenenic differentiation.
Chondrogenesis; Perlecan; Bone Morphogenetic Protein-2 (BMP-2); Collagen II; Mesenchymal Cells; Tissue Engineering
Immobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, in vitro. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF165) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, in vitro.
In solution, PlnDI binds VEGF165 in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF165 mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF165 alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF165 reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951), and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF165 but not VEGF121 significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2.
Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF165 can enhance VEGFR-2 signaling and angiogenic events, in vitro. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, in vivo.
Despite that recombinant human bone morphogenetic protein-2 (rhBMP-2) has been reported as a stimulatory effecter of cancer cell growth because of its characteristic like morphogen, the biological functions of rhBMP-2 in human esophageal cancer cells are unknown. The purpose of this study was to investigate whether rhBMP-2 has an inhibitory effect on the growth of human esophageal squamous carcinoma cells (ESCC). RhBMP-2 significantly inhibited proliferation of ESCC cells in a dose-dependent manner in the MTT assay. Cell cycle arrest at the G1 phase was induced 24 h after rhBMP2 treatment. RhBMP-2 also reduced cyclin D1, cyclin-dependent kinase (CDK) 4 and CDK 6 activities, and stimulated p-Smad1/5/8, p53, and p21 levels at 12 h. In contrast, rhBMP-2 diminished poly (ADP-ribose) polymerase (PARP) protein expression levels and activated cleaved PARP, cleaved caspase-7, and cleaved-caspase 9 levels in ESCC cells. In addition, rhBMP-2 increased MST1, MOB1, and p-YAP protein levels and the RASSF1 binds Mst1 more upon treatment with rhBMP2. The induced p-YAP expression in TE-8 and TE-12 cells by rhBMP-2 was reversed by the RASSF1 knockdown. In vivo study, rhBMP-2 decreased tumor volume following subcutaneous implantation and showed higher radiologic score (less bony destruction) after femoral implantation compared to those in a control group. These results suggest that rhBMP-2 inhibits rather than activates proliferation of human esophageal cancer cells which is mediated through activating the hippo signaling pathway.
The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells. rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of 1 alpha,25- dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type III in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-beta 1 inhibited myogenic differentiation. However, unlike BMP-2, TGF-beta 1 decreased ALP activity in both C26 and C20 cells. TGF-beta 1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP- 2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.
In this study, recombinant human bone morphogenetic protein-2 (rhBMP-2) delivery system with slow mode was successfully developed in three-dimensional (3D) printing-based polycaprolactone (PCL)/poly(lactic-co-glycolic acid) (PLGA) scaffolds for bone formation of critical-sized rabbit segmental diaphyseal defect. To control the delivery of the rhBMP-2, collagen (for long-term delivery up to 28 days) and gelatin (for shor-term delivery within a week) solutions encapsulating rhBMP-2 were dispensed into a hollow cylinderical type of PCL/PLGA scaffold. An effective dose of 5μg/mL was determined by measuring the alkaline phosphatase and osteocalcin gene expression levels of human nasal inferior turbinate-derived mesenchymal stromal cells (hTMSCs) seeded on the PCL/PLGA/collagen scaffold in vitro. However, it was found that a burst release of rhBMP-2 from the PCL/PLGA/gelatin scaffold did not induce the osteogenic differentiation of hTMSCs in vitro at an equivalent dose. In the in vivo animal experiements, microcomputed tomography and histological analyses confirmed that PCL/PLGA/collagen/rhBMP-2 scaffolds (long-term delivery mode) showed the best bone healing quality at both weeks 4 and 8 after implantation without inflammatory response. On the other hand, a large number of macrophages indicating severe inflammation provoked by burst release of rhBMP-2 were observed in the vicinity of PCL/PLGA/gelatin/rhBMP-2 (short-term delivery mode) at week 4.
Recombinant human bone morphogenetic protein 7 (rhBMP7) is applied for treatment of bone fractures, especially tibial non-unions. Its application may induce autoantibodies (aAB) affecting the targeted and endogenous signaling pathways and in turn negatively impact treatment efficacy.
Novel and sensitive assays for the quantification of BMP7-aAB and BMP2-aAB were established and used to analyze serum samples from healthy controls (n = 100 men, n = 100 women) and patients with long bone fracture (n = 265) treated or not with rhBMP7. Sera from three to nine time points per patient were available and enabled the evaluation of aAB over a time course of up to one year. Functional activity of the BMP-aAB was tested with a BMP-responsive cell-based reporter assay. Consolidation of the fracture was evaluated as clinical outcome potentially affected by BMP7-aAB.
Prevalence of BMP7-aAB and BMP2-aAB was 1–2.5% in non-treated patients or healthy controls. The rhBMP7 treatment induced a transient increase in BMP7-aAB in a subset of patients, returning to non-detectable levels within six months. IgG from BMP7-aAB positive sera inhibited dose dependently the BMP7-reporter gene activity, whereas control sera were without effect. Successful consolidation of the fracture was observed in the majority of both aAB-positive and aAB-negative patients.
We conclude that BMP7-aAB can be detected as natural aAB in healthy subjects, and are transiently induced by rhBMP7 therapy in a subset of patients. The aAB are capable of antagonizing BMP7 signaling in vitro, but do not preclude treatment success in patients.
Statement of significance: The treatment of bone fractures with recombinant human BMP7 (rhBMP7) induces a transient peak of autoantibodies to BMP7. These antibodies are active as antagonists in vitro, but do not preclude treatment success. These findings are of relevance to rhBMP7-based treatments and the interpretation of health risks by drug induced autoantibodies.Image 1
•There are patients with natural autoantibodies recognizing BMP7.•In some patients, rhBMP7-therapy induces BMP7 autoantibodies.•BMP7 autoantibodies elicit neutralizing effects on BMP signaling.•Therapy-induced BMP7 autoantibodies disappear over time.•BMP7 autoantibodies seem not to affect therapy success.
Autoimmunity; Consolidation; Biologicals; Fracture; Growth factor; Bone morphogenetic protein
The heparan sulfate proteoglycan, perlecan, is localized to hypertrophic chondrocytes in the growth plates of long bones. Mice mutants for perlecan display severe cartilage and skeletal defects. Previously, we demonstrated that C3H10T1/2 fibroblasts cultured on perlecan stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stain intensely with Alcian blue, and antibodies specific for collagen type II and aggrecan; however, nodules do not express collagen type X, a marker of chondrogenic maturation. In this investigation, we tested the hypothesis that addition of rhBMP-2 to perlecan-induced nodules would promote chondrogenic maturation in vitro. C3H10T1/2 fibroblasts were seeded in Lab-Tek® chambered “Permanox” slides uncoated or coated with perlecan (B&D, 5 μg/well), at a density of 2 × 105 cells/well. The cells were maintained in CMRL-1066 media supplemented with ascorbic acid, citrate, and pyruvate (50 ng/ml). C3H10T1/2 fibroblasts seeded on perlecan-coated wells began to condense and form cell aggregates within 15 min. On the third day postplating, the media was replaced and supplemented with or without rhBMP-2 (50 ng/ml, Genetics Institute®). On day 6 of culture, microscopy revealed that rhBMP-2–treated cultures had significantly proliferated; however, untreated cultures had not. By day 12 of culture, confocal microscopy revealed that perlecan-stimulated nodules treated with rhBMP-2 express a late stage marker of chondrogenesis (collagen type X). Morphologically, cells expressing collagen type X in rhBMP-2–treated nodules appear larger in diameter, relative to cells not expressing collagen type X. Cells cultured on plastic and treated with rhBMP-2 did not form nodules, but attached and spread, yielding a high-density monolayer. In response to rhBMP-2 treatment, these cells also express collagen type X. However, the appearance of collagen type X occurs at a later time point relative to the appearance of collagen type X in perlecan-stimulated nodules. Thus, perlecan-stimulated nodules do mature at a faster rate when treated with rhBMP-2 relative to monolayer cells.
Cartilage; Chondrogenesis; Perlecan; Proteoglycan; rhBMP-2
Temporally and spatially controlled delivery of growth factors in polymeric scaffolds is crucial for engineering composite tissue structures, such as osteochondral constructs. In the present study, microsphere-mediated growth factor delivery in polymer scaffolds and its impact on osteochondral differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Two growth factors, bone morphogenetic protein 2 (rhBMP-2) and insulin-like growth factor I (rhIGF-I), were incorporated as a single concentration gradient or reverse gradient combining two factors in the scaffolds. To assess the gradient making system and the delivery efficiency of polylactic-co-glycolic acid (PLGA) and silk fibroin microspheres, initially an alginate gel was fabricated into a cylinder shape with microspheres incorporated as gradients. Compared to PLGA microspheres, silk microspheres were more efficient in delivering rhBMP-2, probably due to sustained release of the growth factor, while less efficient in delivering rhIGF-I, likely due to loading efficiency. The growth factor gradients formed were shallow, inducing non-gradient trends in hMSC osteochondral differentiation. Aqueous-derived silk porous scaffolds were used to incorporate silk microspheres using the same gradient process. Both growth factors formed deep and linear concentration gradients in the scaffold, as shown by enzyme-linked immunosorbent assay (ELISA). After seeding with hMSCs and culturing for 5 weeks in a medium containing osteogenic and chondrogenic components, hMSCs exhibited osteogenic and chondrogenic differentiation along the concentration gradients of rhBMP-2 in the single gradient of rhBMP-2 and reverse gradient of rhBMP-2/rhIGF-I, but not the rhIGF-I gradient system, confirming that silk microspheres were more efficient in delivering rhBMP-2 than rhIGF-I for hMSCs osteochondrogenesis. This novel silk microsphere/scaffold system offers a new option for the delivery of multiple growth factors with spatial control in a 3D culture environment for both understanding natural tissue growth process and in vitro engineering complex tissue constructs.
silk; fibroin; alginate; polylactic-co-glycolic acid; rhBMP-2; rhIGF-I; gradient; scaffold
Perlecan (Pln) is an abundant heparan sulfate (HS) proteoglycan in the pericellular matrix of developing cartilage, and its absence dramatically disrupts endochondral bone formation. This study examined two previously unexamined aspects of the function of Pln in mesenchymal chondrogenesis in vitro. Using the well established high density micromass model of chondrogenic differentiation, we first examined the requirement for endogenous Pln synthesis and secretion through the use of Pln-targeted ribozymes in murine C3H10T1/2 embryonic fibroblasts. Second, we examined the ability of the unique N-terminal, HS-bearing Pln domain I (PlnDI) to synergize with exogenous bone morphogenetic protein-2 (BMP-2) to support later stage chondrogenic maturation of cellular condensations. The results provide clear evidence that the function of Pln in late stage chondrogenesis requires Pln biosynthesis and secretion, because 60-70% reductions in Pln greatly diminish chondrogenic marker expression in micromass culture. Additionally, these data support the idea that while early chondrocyte differentiation can be supported by exogenous HS-decorated PlnDI, efficient late stage PlnDI supported chondrogenesis requires both BMP-2 and Pln biosynthesis.
perlecan; chondrogenesis; BMP-2; cartilage; heparan sulfate proteoglycan
In this study, we tested the hypothesis that a surface functionalization delivery platform incorporating heparin onto strontium alginate microbeads surfaces would convert this “naive carriers” into “mini-reservoirs” for localized in vivo delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) that will induce functional bone regeneration. In vitro evaluation confirmed that (1) heparin incorporation could immobilize and prolong rhBMP-2 release for approximately 3 weeks; (2) a significant decrease (p<0.01) in rhBMP-2 burst release is attainable depending on initial protein load; and (3) rhBMP-2 released from surface functionalized microbeads retained bioactivity and stimulated higher alkaline phosphatase activity in cultured C2C12 cells when compared with daily administration of fresh bolus rhBMP-2. Subsequently, surface functionalized microbeads were used for in vivo delivery of rhBMP-2 at local sites of posterolateral spinal fusion surgery in rats. The microbeads were loaded into the pores of medical-grade polyepsilone caprolactone-tricalcium phosphate scaffolds before implantation. Results revealed robust bone formation and a biomechanically solid fusion after 6 weeks. When compared with a control group consisting of an equivalent amount of rhBMP-2 that was directly adsorbed onto bare-surfaced microbeads with no heparin, a 5.3-fold increase in bone volume fraction and a 2.6-fold increase in bending stiffness (flexion/extension) were observed. When compared with collagen sponge carriers of rhBMP-2, a 1.5-fold and a 1.3-fold increase in bone volume fraction and bending stiffness were observed, respectively. More importantly, 3D micro-computed tomography images enabled the visualization of a well-contained newly formed bone at ipsilateral implant sites with surface functionalized rhBMP-2 delivery. This was absent with collagen sponge carriers where newly formed bone tissue was poorly contained and crossed over the posterior midline to contralateral implants. These findings are important because of complications with current rhBMP-2 delivery method, including excessive, uncontrolled bone formation.
Recombinant bone morphogenetic protein two (rhBMP2) has been utilised for a variety of clinical applications in orthopaedic surgery and dental procedures. Despite its widespread use, concerns have been raised regarding its short half-life and transient bioactivity in vivo. Recent investigation aimed at developing rhBMP2 synthesized from a shorter polypeptide chain (108 amino acids) has been undertaken.
The osteopromotive properties of BMP2 were investigated on cell behaviour. Five concentrations of rhBMP2_108 including 10, 50, 100, 200 and 500 ng/ml were compared to a commercially available rhBMP2 (100 ng/ml). Each of the working concentrations of rhBMP2_108 were investigated on MC3T3-E1 osteoblasts for their ability to induce osteoblast recruitment, proliferation and differentiation as assessed by alkaline phosphatase (ALP) staining, alizarin red staining, and real-time PCR for genes encoding ALP, osteocalcin (OCN), collagen-1 (COL-1) and Runx2.
The results demonstrate that all concentrations of rhBMP2_108 significantly improved cell recruitment and proliferation of osteoblasts at 5 days post seeding. Furthermore, rhBMP2_108 had the most pronounced effects on osteoblast differentiation. It was found that rhBMP2_108 had over a four fold significant increase in ALP activity at seven and 14 days post-seeding and the concentrations ranging from 50 to 200 ng/ml demonstrated the most pronounced effects. Analysis of real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 further confirmed dose-dependant increases at 14 days post-seeding. Furthermore, alizarin red staining demonstrated a concentration dependant increase in staining at 14 days.
The results from the present study demonstrate that this shorter polypeptide chain of rhBMP2_108 is equally as bioactive as commercially available rhBMP2 for the recruitment of progenitor cells by facilitating their differentiation towards the osteoblast lineage. Future in vivo study are necessary to investigate its bioactivity.
BMP2; Osteoinductive; Osteoinduction; Osteoblast differentiation; Guided bone regeneration
Clinical drawbacks of bone grafting prompt the search for alternative bone augmentation technologies such as use of growth and differentiation factors, gene therapy, and cell therapy. Osteopromotive matrices are frequently employed for the local delivery and controlled release of these augmentation agents. Some matrices also provide an osteoconductive scaffold to support new bone growth. In this study, silkworm-derived silk fibroin was evaluated as an osteoconductive matrix for healing critical sized mid-femoral segmental defects in nude rats. Four treatment groups were assessed over eight weeks: Silk scaffolds (SS) with recombinant human BMP-2 (rhBMP-2) and human mesenchymal stem cells (HMSC) that had been pre-differentiated along an osteoblastic lineage ex vivo (Group I; pdHMSC/rhBMP-2/SS); SS with rhBMP-2 and undifferentiated HMSCs (Group II; udHMSC/rhBMP-2/SS); SS and rhBMP-2 alone (Group III; rhBMP-2/SS); and empty defects (Group IV). Bi-weekly radiographs revealed a progressive and similar increase in Group I–III mean defect mineralization through post-operative week (POW) 8. Radiographs, dual energy x-ray absorptiometry, and micro-computed tomography confirmed that Groups I–III exhibited similar substantial and significantly (p<0.05) greater defect mineralization at POW 8 than the unfilled Group IV defects which remained void of bone. No significant differences in Groups I–III defect healing at POW 8 were apparent using these same assays or mechanical testing. Histology at POW 8 revealed moderately good bridging of the parent diaphyseal cortices with woven and lamellar bone bridging islands of silk matrix in Groups I and III. Group II defects possessed comparatively less new bone which was most abundant adjacent to the parent bone margins. Elsewhere the silk matrix was more often enveloped by poorly differentiated loose fibrous connective tissue. Group IV defects showed minimal new bone formation. None of the treatment groups attained the mean mineralization or the mean biomechanical strength of identical defects implanted with SS and pdHMSCs alone in a previous study. However, addition of rhBMP-2 to SS prompted more bone than was previously generated using udHMSC/SS or SS alone. These data imply the clinical potential of silk scaffolds and rhBMP-2 as composite osteopromotive implants when used alone or with select stem cell populations. Additional studies in larger species are now warranted.
bone healing; silk implant; bone morphogenetic protein; human mesenchymal stem cells; tissue engineering; long bone defect
Bone morphogenetic protein-2 (BMP-2) is considered a promising adjuvant for the treatment of skeletal non-union and spinal fusion. However, BMP-2 delivery in a conventional collagen scaffold necessitates a high dose to achieve an efficacious outcome. To lower its effective dose, we precomplexed BMP-2 with the glycosaminoglycans (GAGs) dermatan sulfate (DS) or heparin (HP), prior to loading it into a hyaluronic acid (HA) hydrogel. In vitro release studies showed that BMP-2 precomplexed with DS or HP had a prolonged delivery compared to without GAG. BMP-2-DS complexes achieved a slightly faster release in the first 24 h than HP; however, both delivered BMP-2 for an equal duration. Analysis of the kinetic interaction between BMP-2 and DS or HP showed that HP had approximately 10 times higher affinity for BMP-2 than DS, yet it equally stabilized the protein, as determined by alkaline phosphatase activity. Ectopic bone formation assays at subcutaneous sites in rats demonstrated that HA hydrogel-delivered BMP-2 precomplexed with GAG induced twice the volume of bone compared with BMP-2 delivered uncomplexed to GAG.
The purpose of this study was to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) with or without osteogenic differentiation medium (ODM) on osteogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBMSCs) in vitro.
The hBMSCs were isolated from medullary reaming tissue. At 80% confluence, hBMSCs were treated with different concentrations of rhBMP-7 with and without ODM. Alkaline phosphatase (ALP) activity, calcium deposition and messenger RNA (mRNA) expression of osteocalcin (OC) and osteopontin (OPN) were examined.
ALP activity and calcium deposits in hBMSC culture were significantly increased by rhBMP-7 at 0.1 μg/ml (0.23 ± 0.07 IU and 28.9 ± 4.2 mg/dl) and 1.0 μg/ml (0.32 ± 0.03 IU and 38.7 ± 3.0 mg/dl), respectively, in the presence of ODM, showing a clearly dose-dependent osteoblastic differentiation. However, the same dose of 0.1 μg/ml rhBMP-7 without ODM and ODM alone induced low level of ALP and calcium deposits, indicating a synergistic effect of rhBMP-7 and ODM on committed osteogenic differentiation. Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed up-regulated OC and OPN mRNA levels, corroborating the synergistic effect of rhBMP-7 and ODM.
Our study showed that rhBMP-7 with ODM created a synergistic effect on up-regulation of osteogenic genes as well as osteogenic differentiation of primary hBMSCs in vitro. In the presence of ODM, the lowest concentration of rhBMP-7 needed to induce significant osteogenic differentiation of hBMSCs was 0.1 μg/ml.