Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells

Acrylated hyaluronic acid (HA) was used as a scaffold for bone morphogenic protein-2 (BMP-2) and human mesenchymal stem cells (hMSCs) for rat calvarial defect regeneration. HA was acrylated by two-step reactions: (1) introduction of an amine group using adipic acid dihydrazide (ADH); (2) acrylation by N-acryloxysuccinimide. Tetrathiolated poly(ethylene) glycol (PEG-SH(4)) was used as a cross-linker by a Michael-type addition reaction and the hydrogel was formed within 10min under physiological conditions. This hydrogel is degraded completely by 100U/ml hyaluronidase in vitro. hMSCs and/or BMP-2 was added during gelation. Cellular viability in vitro was increased up to 55% in the hydrogels with BMP-2 compared with the control. For in vivo calvarial defect regeneration, five different samples (i.e., control, hydrogel, hydrogel with BMP-2, hydrogel with MSCs, and hydrogel with BMP-2 and MSCs) were implanted for 4 weeks. The histological results demonstrated that the hydrogels with BMP-2 and MSCs had the highest expression of osteocalcin and mature bone formation with vascular markers, such as CD31 and vascular endothelial growth factors, compared with the other samples. This study demonstrated that HA base hydrogel can be used for cell and growth factor carriers for tissue regeneration.     

Locally applied vascular endothelial growth factor A increases the osteogenic healing capacity of human adipose-derived stem cells by promoting osteogenic and endothelial differentiation

Human adipose-derived stem cells (hASCs) are known for their capability to promote bone healing when applied to bone defects. For bone tissue regeneration, both sufficient angiogenesis and osteogenesis is desirable. Vascular endothelial growth factor A (VEGFA) has the potential to promote differentiation of common progenitor cells to both lineages. To test this hypothesis, the effects of VEGFA on hASCs during osteogenic differentiation were tested in vitro. In addition, hASCs were seeded in murine critical-sized calvarial defects locally treated with VEGFA. Our results suggest that VEGFA improves osteogenic differentiation in vitro as indicated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time polymerase chain reaction analysis. Moreover, local application of VEGFA to hASCs significantly improved healing of critical-sized calvarial defects in vivo. This repair was accompanied by a striking enhancement of angiogenesis. Both paracrine and, to a lesser degree, cell-autonomous effects of VEGFA-treated hASCs were accountable for angiogenesis. These data were confirmed by using CD31(-) /CD45(-) mouse ASCs(GFP+) cells. In summary, we demonstrated that VEGFA increased osteogenic differentiation of hASCS in vitro and in vivo, which was accompanied by an enhancement of angiogenesis. Additionally, we showed that during bone regeneration, the increase in angiogenesis of hASCs on treatment with VEGFA was attributable to both paracrine and cell-autonomous effects. Thus, locally applied VEGFA might prove to be a valuable growth factor that can mediate both osteogenesis and angiogenesis of multipotent hASCs in the context of bone regeneration.     

Enhanced healing of rat calvarial defects with sulfated chitosan-coated calcium-deficient hydroxyapatite/bone morphogenetic protein 2 scaffolds

Calcium phosphate cements (CPCs), which are widely used in bone regeneration, possess good biocompatibility and osteoconductivity and have been demonstrated to be candidate carriers for bone growth factors. However, limited release of growth factors from CPCs and slow degradation of the materials are not desirable for certain clinical applications. Previous studies have shown that calcium-deficient hydroxyapatite (CDHA) from CPCs presents more rapid degradation rate than CPCs. In this study, a hybrid growth factor delivery system was prepared by using bone morphogenetic protein 2 (BMP-2) loaded CDHA porous scaffold with sulfated chitosan (SCS) coating for improved release profile. We tested the BMP-2 release characteristic of CDHA/BMP-2/SCS composite in vitro and its ability to repair rat calvarial bone defects. A higher percentage of BMP-2 was released when sulfated chitosan coating was present compared with CDHA/BMP-2 group. Eight weeks postoperation, the repaired crania were evaluated by microcomputed tomography, sequential fluorescent labeling, histological analysis, and immunohistochemistry. CDHA/BMP-2/SCS group promoted the most extensive new bone formation than CDHA/BMP-2 and CDHA groups. Our observations suggest that sulfated chitosan coating could enhance the release profile of CDHA/BMP-2 composite in vitro and promote new bone formation in vivo. The hybrid CDHA/BMP-2/SCS system is a promising growth factor delivery strategy for bone regeneration.     

Antibody-mediated osseous regeneration: a novel strategy for bioengineering bone by immobilized anti-bone morphogenetic protein-2 antibodies

Bone regeneration often requires harvesting of autologous bone with significant potential morbidity and cost. Recombinant human bone morphogenetic protein (rhBMP)-2 has been approved by the U.S. Food and Drug Administration for specific regenerative indications. However, administration of exogenous growth factors has many drawbacks. The objective of the present proof-of-concept study was to determine whether immobilized anti-BMP-2 antibodies (Abs) could capture endogenous BMP-2 in local sites to mediate osteogenesis, a strategy we refer to as antibody-mediated osseous regeneration (AMOR). We have generated a murine anti-BMP-2 monoclonal antibody library, which was tested along with commercially available Abs in vitro and in vivo for their ability to mediate AMOR. In vitro studies demonstrated that only some anti-BMP-2 Abs tested formed immune complexes with BMP-2, which can bind to BMP cellular receptor, whereas other BMP-2/anti-BMP-2 complexes failed to bind. To investigate whether anti-BMP-2 Abs were able to mediate AMOR in vivo, anti-BMP-2 Abs were immobilized on absorbable collagen sponge (ACS) and surgically placed in rat calvarial defects. Microcomputed tomography analysis of live animals at 2, 4, and 6 weeks demonstrated that some anti-BMP-2 Abs immobilized on ACS mediated significant bone regeneration, whereas other clones did not mediate any bone regeneration. In situ BMP-2 and osteocalcin expression was investigated by immunohistochemistry. Results demonstrated higher BMP-2 and osteocalcin expression in sites with increased bone regeneration. Results provide first evidence for the ability of anti-BMP2 Abs to form an immune complex with endogenous BMP-2 and mediate bone regeneration in vivo, suggesting a promising therapeutic method for tissue engineering.     

MicroCT evaluation of three-dimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects

Numerous growth factors, peptides, and small molecules are being developed for bone tissue engineering. The optimal dosing, stability, and bioactivity of these biological molecules are likely influenced by the carrier biomaterial. Efficient evaluation of various formulations will require objective evaluation of in vitro culture systems and in vivo regeneration models. The objective of this paper is to examine the utility of microcomputed tomography (microCT) over conventional techniques in the evaluation of the bone morphogenetic protein-2 (BMP-2) dose response effect in a three-dimensional (3D) in vitro culture system and in an established calvarial defect model. Cultured MC3T3-E1 osteoblasts displayed increased cellular density, extracellular matrix (ECM) production, and mineralization on 3D poly(lactic-co-glycolic acid) (PLGA) scaffolds in a BMP-2 dose dependent manner. MicroCT revealed differences in shape and spatial organization of mineralized areas, which would not have been possible through conventional alizarin red staining alone. Additionally, BMP-2 (doses of 30 to 240 ng/mm(3)) was grafted into 5 mm critical sized rat calvarial defects, where increased bone regeneration was observed in a dose dependent manner, with higher doses of BMP-2 inducing greater bone area, volume, and density. The data revealed the utility of microCT analysis as a beneficial addition to existing techniques for objective evaluation of bone tissue engineering and regeneration.     

Dura mater stimulates human adipose-derived stromal cells to undergo bone formation in mouse calvarial defects

Human adipose-derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell-cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM-hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC-DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP-2 and anti-BMP-2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM-hASC interaction dramatically reduced calvarial healing with abrogated BMP-2-Smad-1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP-2 pathway, we found that BMP-2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP-2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC-engrafted calvarial defect. Furthermore, DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC mediated repair.     

Efficient Bone Regeneration Induced by Bone Morphogenetic Protein-2 Released from Apatite-Coated Collagen Scaffolds

Abstract

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. Clinically utilized BMP-2 uses a type-I collagen scaffold as a carrier. Here we hypothesized that an apatite coating on a type-I collagen scaffold would prolong the BMP-2 release period and enhance bone regeneration in calvarial defects in mice. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluid. BMP-2 release kinetics and bioactivity were evaluated by enzyme-linked immunosorbent assay and alkaline phosphatase activity measurement of cultured osteoblasts. Computed tomography and histomorphometry were performed eight weeks after various doses of BMP-2 were delivered to mouse calvarial defects using either non-modified or apatite-coated collagen scaffolds. Apatite-coated collagen scaffolds released 91.8 ± 11.5% of the loaded BMP-2 over 13 days in vitro, whereas non-modified collagen scaffolds released 98.3 ± 2.2% over the initial one day. The in vivo study showed that BMP-2 delivery with apatite-coated collagen scaffolds resulted in a significantly greater bone formation area and higher bone density than that with non-modified collagen scaffolds. This study suggests that simple apatite coating on collagen scaffolds can enhance the bone regeneration efficacy of BMP-2 released from collagen scaffolds.     

Spontaneous Healing Capacity of Calvarial Bone Defects in mdx Mice

The mdx mouse is an experimental model widely used for the study of Duchenne muscular dystrophy, which is characterized by the lack of dystrophin and cycles of muscle degeneration/regeneration. Studies demonstrated elevated levels of growth factors and accelerated skin wound repair in these animals. We therefore raised the hypothesis that the bone repair process might also be altered in these animals. Thus, the objective of this study was to evaluate the spontaneous healing of calvarial defects in mdx mice by histomorphometric analysis. Animals (45 days old) were divided into mdx and control groups. A defect measuring 2 mm in diameter was produced surgically in the right parietal bone of each animal. The animals were sacrificed 15, 30, and 60 days after surgery, and the skulls were processed by routine histological procedures. No difference in the volume of new bone inside the defect was observed between the two groups at any of the three postoperative time points. There was also no difference between the different periods of healing when each group was analyzed separately. The lower quality of femoral and calvarial bone in mdx mice reported in previous studies and the similar bone regeneration rates seen in two groups suggest that the healing capacity of calvarial defects was more expressive in mdx mice than in control animals. An increase in the amount of osteogenic factors released by damaged myofibers may have favored osteogenesis during bone defect healing in mdx mice. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Nell-1-induced bone regeneration in calvarial defects

Many craniofacial birth defects contain skeletal components requiring bone grafting. We previously identified the novel secreted osteogenic molecule NELL-1, first noted to be overexpressed during premature bone formation in calvarial sutures of craniosynostosis patients. Nell-1 overexpression significantly increases differentiation and mineralization selectively in osteoblasts, while newborn Nell-1 transgenic mice significantly increase premature bone formation in calvarial sutures. In the current study, cultured calvarial explants isolated from Nell-1 transgenic newborn mice (with mild sagittal synostosis) demonstrated continuous bone growth and overlapping sagittal sutures. Further investigation into gene expression cascades revealed that fibroblast growth factor-2 and transforming growth factor-beta1 stimulated Nell-1 expression, whereas bone morphogenetic protein (BMP)-2 had no direct effect. Additionally, Nell-1-induced osteogenesis in MC3T3-E1 osteoblasts through reduction in the expression of early up-regulated osteogenic regulators (OSX and ALP) but induction of later markers (OPN and OCN). Grafting Nell-1 protein-coated PLGA scaffolds into rat calvarial defects revealed the osteogenic potential of Nell-1 to induce bone regeneration equivalent to BMP-2, whereas immunohistochemistry indicated that Nell-1 reduced osterix-producing cells and increased bone sialoprotein, osteocalcin, and BMP-7 expression. Insights into Nell-1-regulated osteogenesis coupled with its ability to stimulate bone regeneration revealed a potential therapeutic role and an alternative to the currently accepted techniques for bone regeneration.     

Orthotopic bone formation by implantation of apatite-coated poly(lactide-co-glycolide)/hydroxyapatite composite particulates and bone morphogenetic protein-2

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. However, a delivery system is essential to take advantage of the osteoinductive effect of BMPs. In the present study, we tested the suitability of apatite-coated poly(D,L-lactide-co-glycolide)/nanohydroxyapatite (PLGA/HA) particulates as carriers for the controlled release of BMP-2. The release of BMP-2 from apatite-coated PLGA/HA particulates was sustained for at least 4 weeks in vitro. A delivery system of apatite-coated PLGA/HA particulates suspended in fibrin gel further slowed the BMP-2 release rate. In vivo implantation of either Fibrin gel + BMP-2 or Fibrin gel + apatite-coated PLGA/HA particulates showed enhanced new bone formation in critical-sized calvarial defects of rats 8 weeks after implantation, compared to implantation of fibrin gel only. Importantly, new bone formation was much higher in the defects treated with BMP-2 delivery using apatite-coated PLGA/HA particulates in fibrin gel (Fibrin gel + PLGA/HA + BMP-2 group) than in the defects treated either with apatite-coated PLGA/HA particulates in fibrin gel (Fibrin gel + BMP-2 group) or with BMP-2 delivery using fibrin gel alone (Fibrin gel + BMP-2 group). BMP-2 and osteoinductive HA had an additive effect on orthotopic bone formation. In conclusion, the apatite-coated PLGA/HA particulates showed good results as carriers for BMP-2. The BMP-2 delivery system showed high osteogenic capability in a rat calvarial bone defect model. The local and sustained delivery system for BMP-2 developed in this study may be useful as a carrier for BMP-2 and would enhance bone regeneration efficacy for the treatment of large bone defects.     

Heparin microparticle effects on presentation and bioactivity of bone morphogenetic protein-2

Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration.     

Fgf-18 is required for osteogenesis but not angiogenesis during long bone repair

Bone regeneration is a complex event that requires the interaction of numerous growth factors. Fibroblast growth factor (Fgf)-ligands have been previously described for their importance in osteogenesis during development. In the current study, we investigated the role of Fgf-18 during bone regeneration. By utilizing a unicortical tibial defect model, we revealed that mice haploinsufficient for Fgf-18 have a markedly reduced healing capacity as compared with wild-type mice. Reduced levels of Runx2 and Osteocalcin but not Vegfa accompanied the impaired bone regeneration. Interestingly, our data indicated that upon injury angiogenesis was not impaired in Fgf-18(+/-) mice. Moreover, other Fgf-ligands and Bmp-2 could not compensate for the loss of Fgf-18. Finally, application of FGF-18 protein was able to rescue the impaired healing in Fgf-18(+/-) mice. Thus, we identified Fgf-18 as an important mediator of bone regeneration, which is required during later stages of bone regeneration. This study provides hints on how to engineering efficiently programmed bony tissue for long bone repair.     

Analysis of the roles of microporosity and BMP-2 on multiple measures of bone regeneration and healing in calcium phosphate scaffolds

Osteoinductive agents, such as BMP-2, are known to improve bone formation when combined with scaffolds. Microporosity (<20 μm) has also been shown to influence bone regeneration in calcium phosphate (CaP) scaffolds. However, many studies use only the term "osteoconductive" to describe the effects of BMP-2 and microporosity on bone formation, and do not assess the degree of healing that occurred. The objective of this study was to quantify the influence of BMP-2 and microporosity on bone regeneration and healing in biphasic calcium phosphate scaffolds using multiple measures including bone volume fraction, radial distribution, and specific surface area. These measures were quantitatively compared by analyzing microcomputed tomography data and used to formally define and assess healing. A custom image segmentation program was used to segment >100 samples, with 900 images each, that were implanted in porcine mandibular defects for 3, 6, 12 and 24 weeks. The assessment of healing presented in this work demonstrates the level of detail possible in evaluating scaffold-guided bone regeneration. The analysis shows that BMP-2 and microporosity accelerate healing up to 4-fold. BMP-2 and microporosity were shown to have different and complementary roles in bone formation that effect the time needed for a defect to heal.     

Effects of FGF-2 release from a hydrogel polymer on bone mass and microarchitecture

Bone substitutes are widely used for filling and restoring bone defects. Among them, methacrylic polymers are employed in load-bearing bones to seal hip prostheses. Incorporation of growth factors into a polymer device could be a way to enhance bone growth. In the present study, we evaluated the capacity of poly(2-hydroxyethyl methacrylate) - pHEMA - copolymerized with 2-vinyl pyrrolidone - VP - to release proteins. Fibroblast growth factor-2 (FGF-2) was incorporated into cylinders of p(HEMA-co-VP). FGF-2 release was studied by ELISA in vitro and cylinders were implanted in the femoral condyle of white New Zealand rabbits. After 2 months post-surgery, FGF-2 was able to enhance bone formation by increasing bone volume; this effect was evidenced by an increase in trabecular number and bone gain was mainly in the form of woven bone. At 3 months post-surgery, no difference could be evidenced between animals receiving vehicle or FGF-2. Animals receiving vehicle exhibited bone mass higher than at 2 months and woven bone was replaced by mature bone with a lamellar matrix. The hydrogel polymer allowed the release of FGF-2, which in return enhanced bone regeneration soon after surgery but the effect vanished rapidly.     

Osteogenic differentiation of adipose-derived stem cells and calvarial defect repair using baculovirus-mediated co-expression of BMP-2 and miR-148b

Repair of large calvarial bony defect remains a challenge for orthopedic surgeons. Since microRNAs (miRNAs) modulate the osteogenesis of osteoprogenitor cells, we aimed to engineer human adipose-derived stem cells (hASCs), a promising cell source for bone engineering, with miRNA-expressing baculovirus vectors. We constructed 4 baculoviruses each expressing 1 human miRNA (miR-26a, miR-29b, miR-148b, miR-196a) and verified that the miRNA-expressing baculovirus vectors augmented hASCs osteogenesis. Among these 4 miRNAs, miR-148b and miR-196a exerted more potent osteoinductive effects than miR-26a and miR-29b. Furthermore, we unveiled that co-transduction of hASCs with miR-148b-expressing and bone morphogenetic protein 2 (BMP-2)-expressing baculovirus vectors enhanced and prolonged BMP-2 expression, and synergistically promoted the in vitro osteogenic differentiation of hASCs. Implantation of the hASCs co-expressing BMP-2/miR-148b into critical-size (4 mm in diameter) calvarial bone defects in nude mice accelerated and potentiated the bone healing and remodeling, filling ≈94% of defect area and ≈89% of defect volume with native calvaria-like flat bone in 12 weeks, as judged from micro computed tomography, histology and immunohistochemical staining. Altogether, this study confirmed the feasibility of combining miRNA and growth factor expression for synergistic stimulation of in vitro osteogenesis and in vivo calvarial bone healing.     

Functionalization of scaffolds with chimeric anti-BMP-2 monoclonal antibodies for osseous regeneration

Recent studies have demonstrated the ability of murine anti-BMP-2 monoclonal antibodies (mAb) immobilized on an absorbable collagen sponge (ACS) to mediate de novo bone formation, a process termed antibody-mediated osseous regeneration (AMOR). The objectives of this study were to assess the efficacy of a newly generated chimeric anti-BMP-2 mAb in mediating AMOR, as well as to evaluate the suitability of different biomaterials as scaffolds to participate in AMOR. Chimeric anti-BMP-2 mAb was immobilized on 4 biomaterials, namely, titanium microbeads (Ti), alginate hydrogel, macroporous biphasic calcium phosphate (MBCP) and ACS, followed by surgical implantation into rat critical-size calvarial defects. Animals were sacrificed after 8 weeks and the degree of bone fill was assessed using micro-CT and histomorphometry. Results demonstrated local persistence of chimeric anti-BMP-2 mAb up to 8 weeks, as well as significant de novo bone regeneration in sites implanted with chimeric anti-BMP-2 antibody immobilized on each of the 4 scaffolds. Ti and MBCP showed the highest volume of bone regeneration, presumably due to their resistance to compression. Alginate and ACS also mediated de novo bone formation, though significant volumetric shrinkage was noted. In vitro assays demonstrated cross-reactivity of chimeric anti-BMP-2 mAb with BMP-4 and BMP-7. Immune complex of anti-BMP-2 mAb with BMP-2 induced osteogenic differentiation of C2C12 cells in vitro, involving expression of RUNX2 and phosphorylation of Smad1. The present data demonstrated the ability of chimeric anti-BMP-2 mAb to functionalize different biomaterial with varying characteristics to mediate osteogenesis.     

Enhancing bioactivity of chitosan film for osteogenesis and wound healing by covalent immobilization of BMP-2 or FGF-2

This study compares the efficacy of growth factors that are covalently immobilized to those that are adsorbed in improving the bioactivity of a biomaterial. Bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) was covalently bonded to chitosan films using carbodiimide chemistry. For BMP-2, a growth factor loading efficiency of ～64% was obtained with this method compared to ～25% from adsorption. As for FGF-2, the growth factor loading efficiency of the two methods was similar at ～50%. The covalently immobilized BMP-2 promoted attachment, proliferation, and differentiation of osteoblasts in a dose-dependent manner, whereas the covalently immobilized FGF-2 stimulated fibroblast attachment, proliferation, and collagen synthesis. After three?weeks immersion in phosphate buffered saline, about 80% of the covalently immobilized growth factors were retained on the films, while only ～16 and ～21% of the adsorbed BMP-2 and FGF-2 remained on the corresponding films. The higher retention rate of the covalently immobilized growth factors enabled their stimulatory effects to persist for a longer period than when adsorbed growth factors were used.     

Bone regeneration using an alpha 2 beta 1 integrin-specific hydrogel as a BMP-2 delivery vehicle

Non-healing bone defects present tremendous socioeconomic costs. Although successful in some clinical settings, bone morphogenetic protein (BMP) therapies require supraphysiological dose delivery for bone repair, raising treatment costs and risks of complications. We engineered a protease-degradable poly(ethylene glycol) (PEG) synthetic hydrogel functionalized with a triple helical, α2β1 integrin-specific peptide (GFOGER) as a BMP-2 delivery vehicle. GFOGER-functionalized hydrogels lacking BMP-2 directed human stem cell differentiation and produced significant enhancements in bone repair within a critical-sized bone defect compared to RGD hydrogels or empty defects. GFOGER functionalization was crucial to the BMP-2-dependent healing response. Importantly, these engineered hydrogels outperformed the current clinical carrier in repairing non-healing bone defects at low BMP-2 doses. GFOGER hydrogels provided sustained in vivo release of encapsulated BMP-2, increased osteoprogenitor localization in the defect site, enhanced bone formation and induced defect bridging and mechanically robust healing at low BMP-2 doses which stimulated almost no bone regeneration when delivered from collagen sponges. These findings demonstrate that GFOGER hydrogels promote bone regeneration in challenging defects with low delivered BMP-2 doses and represent an effective delivery vehicle for protein therapeutics with translational potential.     

Bioluminescent and micro-computed tomography imaging of bone repair induced by fibrin-binding growth factors

In this work we have evaluated the capacity of bone morphogenetic protein-2 (BMP-2) and fibrin-binding platelet-derived growth factor-BB (PDGF-BB) to support cell growth and induce bone regeneration using two different imaging technologies to improve the understanding of structural and organizational processes participating in tissue repair. Human mesenchymal stem cells from adipose tissue (hAMSCs) expressing two luciferase genes, one under the control of the cytomegalovirus (CMV) promoter and the other under the control of a tissue-specific promoter (osteocalcin or platelet endothelial cell adhesion molecule), were seeded in fibrin matrices containing BMP-2 and fibrin-binding PDGF-BB, and further implanted intramuscularly or in a mouse calvarial defect. Then, cell growth and bone regeneration were monitored by bioluminescence imaging (BLI) to analyze the evolution of target gene expression, indicative of cell differentiation towards the osteoblastic and endothelial lineages. Non-invasive imaging was supplemented with micro-computed tomography (μCT) to evaluate bone regeneration and high-resolution μCT of vascular casts. Results from BLI showed hAMSC growth during the first week in all cases, followed by a rapid decrease in cell number; as well as an increment of osteocalcin but not PECAM-1 expression 3 weeks after implantation. Results from μCT show that the delivery of BMP-2 and PDGF-BB by fibrin induced the formation of more bone and improves vascularization, resulting in more abundant and thicker vessels, in comparison with controls. Although the inclusion of hAMSCs in the fibrin matrices made no significant difference in any of these parameters, there was a significant increment in the connectivity of the vascular network in defects treated with hAMSCs.