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.
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
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.
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
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
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
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.
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.
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
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.
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
The expression patterns of (bone morphogenetic proteins) BMPs during fracture repair and pre-natal bone development suggests that these processes are regulated through the coordinated actions of multiple BMPs. Murine bone marrow stromal cells (MSCs) in culture provide a well recognized ex vivo system of mesenchymal stem cell differentiation in which the effects of BMPs can be examined. Studies were performed to determine if MSC differentiation is dependent on the endogenous expression of multiple BMPs and to characterize their interactions. MSCs were harvested from the bone marrow of tibiae and femora of 8 to 10 week old male C57/B6 mice and prepared by standard methods. Osteogenic differentiation was assessed by histological assays, alkaline phosphatase enzyme activity and assays for the expression of multiple mRNAs for BMPs and osteogenic development. The role of autogenously expressed BMPs in controlling the osteogenic differentiation of marrow stromal cells in vitro was assessed in both gain-of-function and loss-of-function experiments. Gain of function experiments were carried out in the presence of exogenously added BMP-2 or 7 and loss of function experiments were carried out by BMP antagonism with noggin and BMP-2 antibody blockade. Osteogenic differentiation was concurrent with and proportional to increases in the expression of BMPs 2, 3, 4, 5, 6, and 8A. BMP antagonism with either noggin or BMP-2 antibody blockade inhibited osteogenic differentiation by 50% to 80% respectively and reduced the expression of endogenous levels of BMPs 2, 3, 5, and 8A. In contrast, antagonism induced the expression of BMP-4 and 6. The addition of rhBMP-2 or 7 enhanced osteogenic differentiation and produced a reciprocal expression profile in the endogenous BMPs expression as compared to BMP antagonism. BMP antagonism could be rescued through the competitive addition of rhBMP-2. These studies demonstrated that osteogenic differentiation was regulated by a complex network of multiple BMPs that showed selective increased and decreased expression during differentiation. They further demonstrated that BMP-2 was a central regulator in this network.
Marrow Stromal Stem Cells; Bone Morphogenetic Proteins; BMP; Osteoinduction; Noggin
Pharmacological glucocorticoids (GCs) inhibit bone formation, leading to osteoporosis. GCs inhibit bone morphogenetic protein-2 (Bmp2) expression, and rhBMP-2 restores mineralization in GC-arrested osteoblast cultures. To better understand how GCs regulate BMPs, we investigated Bmp transcription, as well as rhBMP-induced Smad and alkaline phosphatase (ALP) activity. Bmp2 cis-regulatory regions were analyzed by reporter plasmids and LacZ-containing bacterial artificial chromosomes. We found that GCs inhibited Bmp2 via a domain >50 kb downstream of the coding sequence. Bmp expression was evaluated by RT-PCR; whereas GCs strongly inhibited Bmp2, Bmp4 was abundantly expressed and resistant to GCs. Both rhBMP-2 and rhBMP-4 restored mineralization in GC-arrested cultures; rhBMP-2 was 5-fold more effective when dosing was based on ALP activation, however, the rhBMPs were equipotent when dosing was based on Smad transactivation. In conclusion, GCs regulate Bmp2 via a far-downstream domain, and activation of Smad, not ALP, best predicts the pro-mineralization potential of rhBMPs.
Osteoblast; BMP; Smad; alkaline phosphatase; glucocorticoids
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.
Bone defects and nonunions are major clinical skeletal problems. Growth factors are commonly used to promote bone regeneration; however, the clinical impact is limited because the factors do not last long at a given site. The introduction of tissue engineering aimed to deter the diffusion of these factors is a promising therapeutic strategy. The purpose of the present study was to evaluate the in vivo osteogenic capability of an engineered bone morphogenetic protein-4 (BMP4) fusion protein.
BMP4 was fused with a nanosized carrier, collagen-binding domain (CBD), derived from fibronectin. The stability of the CBD-BMP4 fusion protein was examined in vitro and in vivo. Osteogenic effects of CBD-BMP4 were evaluated by computer tomography after intramedullary injection without a collagen–sponge scaffold. Recombinant BMP-4, CBD, or vehicle were used as controls. Expressions of bone-related genes and growth factors were compared among the groups. Osteogenesis induced by CBD-BMP4, BMP4, and CBD was also assessed in a bone-defect model.
In vitro, CBD-BMP4 was retained in a collagen gel for at least 7 days while BMP4 alone was released within 3 hours. In vivo, CBD-BMP4 remained at the given site for at least 2 weeks, both with or without a collagen–sponge scaffold, while BMP4 disappeared from the site within 3 days after injection. CBD-BMP4 induced better bone formation than BMP4 did alone, CBD alone, and vehicle after the intramedullary injection into the mouse femur. Bone-related genes and growth factors were expressed at higher levels in CBD-BMP4-treated mice than in all other groups, including BMP4-treated mice. Finally, CBD-BMP4 potentiated more bone formation than did controls, including BMP4 alone, when applied to cranial bone defects without a collagen scaffold.
Altogether, nanocarrier-CBD enhanced the retention of BMP4 in the bone, thereby promoting augmented osteogenic responses in the absence of a scaffold. These results suggest that CBD-BMP4 may be clinically useful in facilitating bone formation.
BMP4; bone repair; bone tissue engineering; osteogenesis
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.
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
Poly(ε-caprolactone fumarate) (PCLF) scaffold formulations were assessed as a delivery system of recombinant human bone morphogenetic protein (rhBMP-2) for bone tissue engineering. The formulations included PCLF with combinations of poly(vinyl alcohol) (PVA) and hydroxyapatite (HA). The assessments included in vitro and in vivo assays. In vitro assays validated cell attachment using a pre-osteoblast cell line (MC3T3-E1). Additionally, in vitro release profiles of rhBMP-2 from PCLF scaffolds were determined up to 21 days. Data suggested PCLF incorporated with PVA and HA accelerated rhBMP-2 release and the released protein was bioactive. For the in vivo study, a critical sized defect (CSD) model in a rabbit calvaria was used to test PCLF scaffolds. At 6 weeks post-implantation, significantly more bone formation was measured in PCLF scaffolds containing rhBMP-2 than in scaffolds without rhBMP-2. In conclusion, we demonstrated PCLF delivered biologically active rhBMP-2, promoted bone healing in a CSD and has potential as a bone tissue engineering scaffold.
poly(ε-caprolactone fumarate); three-dimensional scaffold; rabbit calvarial critical sized defect; rhBMP-2; bone tissue engineering
Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.
heparan sulfate proteoglycan; perlecan; chondrogenesis
Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity.
Bone morphogenetic proteins; thrombospondin 4; tendon markers
Functionalized biodegradable nanoparticles (NPs) provide reactive groups and large surface area for grafting Recombinant human bone morphogenetic protein-2 (rhBMP-2) to reduce protein diffusion and maintain sufficient concentration for recruitment and differentiation of osteoprogenitor cells. The objective of this work was to investigate release characteristics and osteogenic activity of rhBMP-2, grafted to biodegradable NPs based on succinimide-terminated poly(lactide fumarate) (PLAF-NHS) and poly(lactide-co-glycolide fumarate) (PLGF-NHS) macromers. The release of rhBMP-2 from the NPs, measured by enzyme-linked immunosorbent assay, was linear with time in the first two weeks, and 24.70±1.30% and 48.7±0.7% of the protein grafted to PLGF-NHS and PLAF-NHS NPs, respectively, was released in the enzymatically active conformation after complete degradation/erosion of the NPs. After 14 days of incubation with bone marrow stromal (BMS) cells, rhBMP-2 grafted to PLAF-NHS and PLGF-NHS NPs was as effective in inducing mineralization as the native rhBMP-2 that was directly added to the cell culture media. At any incubation time, rhBMP-2 grafted to PLAF had the highest expression of osteopontin (OP) and osteocalcin (OC), followed by rhBMP-2 grafted to PLGF and rhBMP-2 directly added to media. Higher OP and OC expression for BMP-gPLAF and BMP-gPLGF groups may be related to other factors in the cascade of osteogenesis, such as differentiation of BMS cells to the vasculogenic lineage and formation of a vascularized/mineralized marix.
Perlecan (Pln) is a large proteoglycan that can bear HS (heparan sulfate) and chondroitin sulfate glycosaminoglycans. Previous studies have demonstrated that Pln can interact with growth factors and cell surfaces either via its constituent glycosaminoglycan chains or core protein. Herein, we summarize studies demonstrating spatially and temporally regulated expression of Pln mRNA and protein in developing and mature cartilage. Mutations either in the Pln gene or in genes involved in glycosaminoglycan assembly result in severe cartilage phenotypes seen in both human syndromes and mouse model systems. In vitro studies demonstrate that Pln can trigger chondrogenic differentiation of multipotential mouse CH310T1/2 stem cells as well as maintain the phenotype of adult human chondrocytes. Structural mapping indicates that these activities lie entirely within domain I, a region unique to Pln, and that they require glycosaminoglycans. We also discuss data indicating that Pln cooperates with the key chondrogenic growth factor, BMP-2, to promote expression of hypertrophic chondrocyte markers. Collectively, these studies indicate that Pln is an important component of human cartilage and may have useful applications in tissue engineering and cartilage-directed therapeutics.
Perlecan; Heparan Sulfate Proteoglycan; Extracellular Matrix; Cartilage; Mouse
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.
Bone grafting procedures have become common due in part to a global trend of population aging. Native bone graft is a popular choice when compared to various synthetic bone graft substitutes, owing to superior biological activity. Nonetheless, the insufficient ability of bone allograft to induce new bone formation and the insufficient remodeling of native bone grafts call for osteoinductive factors during bone repair, exemplified by recombinant human bone morphogenetic protein 2 (rhBMP2). We previously developed a modular bone morphogenetic peptide (mBMP) to address complications associated with the clinical use of rhBMP2 as a bone graft substitute. The mBMP is designed to strongly bind to hydroxyapatite, the main inorganic component of bone and teeth, and to provide pro-osteogenic properties analogous to rhBMP2. Our previous in vivo animal studies showed that mBMP bound to hydroxyapatite-coated orthopedic implants with high affinity and stimulated new bone formation. In this study, we demonstrate specific binding of mBMP to native bone grafts. The results show that mBMP binds with high affinity to both cortical and trabecular bones, and that the binding is dependent on the mBMP concentration and incubation time. Importantly, efficient mBMP binding is achieved in an ex vivo bone bioreactor where bone tissue is maintained viable for several weeks. In addition, mBMP binding can be localized with spatial control on native bone tissue via simple methods, such as dip-coating, spotting and direct writing. Taken together with the pro-osteogenic activity of mBMP established in previous bone repair models, these results suggest that mBMP may promote bone healing when coated on native bone grafts in a clinically compatible manner.
bone morphogenetic protein; synthetic peptide; bone graft; binding
Bone grafts are widely used in orthopaedic procedures. Autografts are limited by donor site morbidity while allografts are known for considerable infection and failure rates. A synthetic composite bone graft substitute poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) was previously developed to stably press-fit in and functionally repair critical-sized rat femoral segmental defects when it was preabsorbed with a single low dose of 300 ng recombinant human bone morphogenetic protein-2/7 (rhBMP-2/7).
To facilitate clinical translation of pHEMA-nHA as a synthetic structural bone graft substitute, we examined its ability to encapsulate and release rhBMP-2 and the antibiotic vancomycin.
We analyzed the compressive behavior and microstructure of pHEMA-nHA as a function of vancomycin incorporation doses using a dynamic mechanical analyzer and a scanning electron microscope. In vitro release of vancomycin was monitored by ultraviolet-visible spectroscopy. Release of rhBMP-2 from pHEMA-nHA-vancomycin was determined by ELISA. Bioactivity of the released vancomycin and rhBMP-2 was examined by bacterial inhibition and osteogenic transdifferentiation capabilities in cell culture, respectively.
Up to 4.8 wt% of vancomycin was incorporated into pHEMA-nHA without compromising its structural integrity and compressive modulus. Encapsulated vancomycin was released in a dose-dependent and sustained manner in phosphate-buffered saline over 2 weeks, and the released vancomycin inhibited Escherichia coli culture. The pHEMA-nHA-vancomycin composite released preabsorbed rhBMP-2 in a sustained manner over 8 days and locally induced osteogenic transdifferentiation of C2C12 cells in culture.
pHEMA-nHA can encapsulate and deliver vancomycin and rhBMP-2 in a sustained and localized manner with reduced loading doses.
The elasticity, osteoconductivity, and rhBMP-2/vancomycin delivery characteristics of pHEMA-nHA may benefit orthopaedic reconstructions or fusions with enhanced safety and efficiency and reduced infection risk.