Biodegradable composites with antibiotics and growth factors for dual release kinetics.

Bone infections are still a major problem in surgery. To avoid severe side effects of systemically administered antibiotics, local antibiotic therapy is increasingly being considered. Using a pressure-based method developed in our group, microporous ß-TCP ceramics, which had previously been characterized, were loaded with 2% w/v alginate containing 50 mg/mL clindamycin and 10 µg/mL rhBMP-2. Release experiments were then carried out over 28 days with changes of liquid at defined times (1, 2, 3, 6, 9, 14, 21 and 28d). The released concentrations of clindamycin were determined by HPLC and those of rhBMP-2 by ELISA. Continuous release (anomalous transport) of clindamycin and uniform release (Fick's diffusion) of BMP-2 were determined. The composites were biocompatible (live/dead, WST-I and LDH) and the released concentrations were all antimicrobially active against Staph. aureus. The results were very promising and clindamycin was detected in concentrations above the MIC as well as a constant rhBMP-2 release over the entire study period. Biocompatibility was also not impaired by either the antibiotic or the BMP-2. This promising approach can therefore be seen as an alternative to the common treatment with PMMA chains containing gentamycin, as the new composite is completely biodegradable and no second operation is necessary for removal or replacement.

Hyaluronic acid-coated gold nanorods enhancing BMP-2 peptide delivery for chondrogenesis.

Bone morphogenic protein-2 (BMP-2) knuckle epitope peptide has been recently discovered and known to activate chondrogenesis. However, the applications of this soluble peptide remain very limited due to rapid diffusion resulting in poor cellular uptake into target cells. We herein designed nanoparticles made from hyaluronic acid functionalized gold nanorods (GNRs) to conjugate with thiolated BMP-2 knuckle epitope peptide via a two-step reaction. Hyaluronic acid was modified to have thiol functional groups to replace the cetyl trimethylammonium bromide ligands on the surface of GNRs. The thiolated peptides were subsequently reacted with hyaluronic acid on the surface on GNRs via a maleimide-hydrazide crosslinker. The conjugation was confirmed by the change of surface charge of GNRs and the plasmon shift. A colorimetric peptide assay suggested more than 69% of the thiolated peptides were conjugated with the hyaluronic acid coated gold nanorods. Moreover, in vitro cell viability showed that BMP-2 conjugated hyaluronic acid functionalized gold nanorods (B2HGR) were cytocompatible and did not cause cytotoxicity to fibroblast cells. The B2HGRs also significantly promote cellular uptake of the BMP-2 peptides in both human mesenchymal stem cells and porcine chondrocytes due to multivalent ligand binding to the BMP receptors on the cell surface resulting in receptor-mediated endocytosis. The enhanced cellular uptake was clearly observed under a confocal microscope resulting in the significant activation of type II collagen gene expression and glucosaminoglycan secretion in those cells. Furthermore, our delivery system is a proof-of-concept of using scaffolds in combination with nanodelivery platform to enhance cartilaginous repair. The peptide loading capacity and the release is not limited by the scaffolds. Therefore, our delivery platform has potential applications for cartilage regeneration in a preclinical and clinical setting in the future.

Effect of Morphogenetic Protein BMP-2 on X-Ray Density of Bone Defect in the Experiment.

In experiments on Wistar rats, a simulated defect in the flat bones of the skull was filled with a collagen sponge of animal origin impregnated with BMP-2 or pure sponge; in control rats, the defect was left open. During follow-up, X-ray density of the collagen sponge in the experimental groups differed significantly. The results attest to the absence of spontaneous remodeling of the bone tissue under conditions modeled focal defect. Moreover, stimulation of reparative processes by the collagen matrix did not lead to positive dynamics. Saturation of the collagen sponge with BMP-2 in a concentration of 0.05 mg/ml allowed increasing Xray density of the bone starting from week 4.

Growth factor sustained delivery from poly-lactic-co-glycolic acid microcarriers and its mass transfer modeling by finite element in a dynamic and static three-dimensional environment bioengineered with stem cells.

Poly-lactic-co-glycolic acid (PLGA) microcarriers (0.8 ± 0.2 µm) have been fabricated with a load of 20 µg/gPLGA by an emulsion-based-proprietary technology to sustained deliver human bone morphogenetic protein 2 (hBMP2), a growth factor largely used for osteogenic induction. hBMP2 release profile, measured in vitro, showed a moderate "burst" release of 20% of the load in first 3 days, followed by a sustained release of 3% of the load along the following 21 days. PLGA microbeads loaded with fluorescent marker (8 mg/gPLGA ) and hydroxyapatite (30 mg/gPLGA ) were also fabricated and successfully dispersed within three-dimensional (3D) alginate scaffold (Ca-alginate 2% wt/wt) in a range between 50 and 200 mg/cm3 ; the presence of microcarriers within the scaffold induced a variation of its stiffness between 0.03 and 0.06 MPa; whereas the scaffold surface area was monitored always in the range of 190-200 m2 /g. Uniform microcarriers dispersion was obtained up to 200 mg/cm3 ; higher loading values in the 3D scaffold produced large aggregates. The release data and the surface area were, then, used to simulate by finite element modeling the hBMP2 mass transfer within the 3D hydrogel bioengineered with stem cells, in dynamic and static cultivations. The simulation was developed with COMSOL Multiphysics® giving a good representation of hBMP2 mass balances along microbeads (bulk eroded) and on cell surface (cell binding). hBMP2 degradation rate was also taken into account in the simulations. hBMP2 concentration of 20 ng/cm3 was set as a target because it has been described as the minimum effective value for stem cells stimulation versus the osteogenic phenotype. The sensitivity analysis suggested the best microbeads/cells ratio in the 3D microenvironment, along 21 days of cultivations in both static and dynamic cultivation (perfusion) conditions. The simulated formulation was so assembled experimentally using human mesenchymal stem cells and an improved scaffold stiffness up to 0.09 MPa (n = 3; p ≤ 0.01) was monitored after 21 days of cultivation; moreover a uniform extracellular matrix deposition within the 3D system was detected by Von Kossa staining, especially in dynamic conditions. The results indicated that the described tool can be useful for the design of 3D bioengineered microarchitecture by quantitative understanding.

In Vitro Osteogenic Differentiation Enhanced by Zirconia Coated with Bone Morphogenetic Protein-2.

In this study we report on the effectiveness of click chemistry-enhanced zirconium dioxide (ZrO2-3) for the immobilization of biomolecules, and the enhancement of osteoblastic differentiation of MC3T3-E1 cells by bone morphogenetic protein-2 (BMP-2) immobilized on ZrO2-6. The surfaces of ZrO2-1 through 6 were characterized by scanning electron microscopy (SEM), static contact angles, and X-ray photoelectron spectroscopy (XPS) measurements. The results from these tests indicated that ZrO2-1 was successfully surface-modified via click chemistry (ZrO2-3). Through quantitative analysis of heparin immobilized on ZrO2-5, we found that ZrO2-3 was a useful tool for immobilizing biomolecules such as heparin. Release tests of BMP-2 from ZrO2-6 showed well-controlled release kinetics over a period of 28 days. MC3T3-E1 cell proliferation tests indicated that ZrO2-6 was highly biocompatible with these cells. Through In Vitro tests such as alkaline phosphatase (ALP) activity, calcium deposition, and real-time polymerase chain reaction (real-time PCR), we found that ZrO2-6 was a useful tool for enhancing osteoblastic differentiation of MC3T3-E1 cells.

[A novel tissue-engineered bone constructed by using human adipose-derived stem cells and biomimetic calcium phosphate scaffold coprecipitated with bone morphogenetic protein-2].

OBJECTIVE: To construct a novel biomimetic calcium phosphate (BioCaP) scaffold loaded with bone morphogenetic protein-2 (BMP-2), and to investigate its role in the osteogenesis of human adipose-derived stem cells (hASCs) in vitro and in vivo. METHODS: The BioCaP scaffold coprecipitated with BMP-2 (BMP-2-BioCaP) was constructed in this study. Field emission scanning electron microscopy (SEM) was used to analyze the morphology of the surfaces. The release kinetics was measured to evaluate the slow-release characteristics in vitro. BMP-2-BioCaP was immersed in proliferation medium (PM) or osteogenic medium (OM), respectively. The supernatants were collected and used to culture hASCs in vitro. Cell numbers were determined using the cell-counting kit-8 (CCK-8) to assess the cell proliferation. After 7 and 14 days, alkaline phosphatase (ALP) staining and quantification were performed to test the activity of ALP. After 14 and 21 days, the calcification deposition was determined by alizarin red S (ARS) staining and quantification. The expressions of the osteoblast-related genes were tested on day 4 and day 14. In the in vivo study, 6 nude mice were used and implanted subcutaneously into the back of the nude mice for 4 groups: (1) BioCaP scaffold only, (2) BioCaP scaffold+hASCs, (3) BMP-2-BioCaP scaffold, (4) BMP-2-BioCaP scaffold+hASCs (test group). After 4 weeks of implantation, hematoxylin-eosin (HE) staining was performed to evaluate the in vivo osteogenesis of hASCs. RESULTS: SEM observations showed that BioCaP and BMP-2-BioCaP scaffold were entirely composed of straight, plate-like and sharp-edged crystal units, and the length of the crystal units varied between 5 and 10 µm. Release kinetics analysis demonstrated that BMP-2 incorporated with BioCaP could be released at certain concentration and last for more than 21 days, and the accumulative protein release could reach 20%. CCK-8 assays showed that cell proliferation was not significantly affected by BMP-2-BioCaP. ALP activity was higher by the induction of OM+BMP-2-BioCaP than of the other groups (P<0.01). More mineralization deposition and more expressions of osteoblast-related genes such as Runt-related transcription factor 2 (RUNX2), ALP, osteopontin (OPN) and osteocalcin (OC) were determined in the OM+BMP-2-BioCaP group at different time points (P<0.01). HE staining showed that, in the test group and BMP-2-BioCaP scaffold group, the extracellular matrix (ECM) with eosinophilic staining were observed around hASCs, and newly-formed bone-like tissues could be found in ECM around the scaffold materials. Moreover, compared with the BMP-2-BioCaP scaffold group, more bone-like tissues could be observed in ECM with typical structure of bone tissue in the test groups. No obvious positive results were found in the other groups. CONCLUSION: BMP-2-BioCaP scaffold could achieve slow-release of BMP-2 and promote the osteogenic differentiation of hASCs in vitro and in vivo. The novel tissue-engineered bone composed of hASCs and BMP-2-BioCaPis promising for the repair of bone defect.

[Synthesis and in vitro characterization of chitosan microspheres/ceramic bovine bone composite scaffold].

OBJECTIVE: The chitosan microspheres encapsulated with bone morphogenetic protein-2 (BMP-2) were prepared by the emulsion cross-linking method. Then the chitosan microspheres were loaded in the ceramic bovine bone (CBB) to achieve the drug delivery system. METHODS: The chemical structure and surface morphology of the drug delivery system were investigated by Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM) observation. Characterization preserved the loading capacity and encapsulation efficiency of the BMP-2. The dynamic immersion method was used to examine the in vitro release characteristic of BMP-2. RESULTS: The chitosan microspheres were successfully encapsulated BMP-2 by cross-linking method. The microspheres were micron-sized (5.982 µm) and spherical in shape with smooth surface morphology. From the release experiments, it was found that 5 mg chitosan/BMP-2 soaked for 21 days with a gradual release of BMP-2. The concentration of BMP-2 was (239.1±20.0) mg/L on Day 21. The in vitro experiment showed that this novel drug delivery system could accelerate MC3T3-E1 cells proliferation and osteogenic differentiation. CONCLUSION: The drug delivery system achieves the biological function of BMP-2 and sustaining slow release in bone repair parts. Also it can provide the basis for repair of bone tissue defect treatment and the selection of bone tissue engineering scaffolds.

Pre-clinical evaluation of the osteogenic potential of bone morphogenetic protein-2 loaded onto a particulate porcine bone biomaterial.

AIM: The objective of study was to determine the osteogenic potential of bone morphogenetic protein-2 (BMP-2) loaded onto a particulate porcine bone mineral (PBM) biomaterial using a sinus augmentation model. METHODS: Release kinetics of BMP-2/PBM was determined in vitro. Eight rabbits received BMP-2/PBM or PBM alone into contra-lateral sinus sites. The animals were killed following a 2-week healing interval for micro-CT and histometrical analysis. RESULTS: Approximately 40% of the BMP-2 was released from PBM over the first 3 days in vitro; release maintained at a reduced level through day 21. In vivo, total augmented implant volume did not differ significantly between treatments. However, local bone formation was enhanced in the BMP-2/PBM group compared with PBM control (10.5% versus 6.6%; p = 0.03), specifically in the central aspect of the PBM implant (14.2% versus 5.5%; p < 0.01) and adjoining the Schneiderian membrane (11.9% versus 5.0%; p < 0.05). There were no significant overall differences in residual biomaterial and fibrovascular tissue. CONCLUSION: Bone morphogenetic protein-2 enhanced local bone formation in the rabbit maxillary sinus model following implantation using a PBM carrier.

Pre-incubation of chemically crosslinked hyaluronan-based hydrogels, loaded with BMP-2 and hydroxyapatite, and its effect on ectopic bone formation.

The effects of pre-incubation of hyaluronan hydrogels, for different lengths of time after the initiation of chemical crosslinking and prior to injection, were explored both by investigating the in vitro BMP-2 release kinetics from the hydrogel and by studying the ectopic bone formation in rats. From the curing profile, obtained from rheological analysis, appropriate pre-incubation times (1 min, 5 h and 3 days) were selected, to prepare slightly, moderately and fully cured hydrogels. Comparable release profiles were observed for all three test groups in vitro. Furthermore, radiography, pQCT and histology of the explanted grafts showed cancellous bone formation in all groups after 5 weeks in vivo. However, longer pre-incubation times gave rise to an increase in bone volume, but a decrease in bone density. Moreover, the 5 h and the 3 days grafts appeared to be more ordered and resistant to deformation from the surrounding tissue than the 1 min grafts. The observed variations in mechanical and biological properties could potentially be used to adapt the treatment for a specific indication.

The short-term effects of repetitive E. coli-derived rhBMP-2 administration through intravenous injection in rats.

Escherichia coli-derived recombinant human (rh)BMP-2 (E.BMP-2) can be used as a bone graft substitute because to its high osteoinductivity, but its toxicity is not well understood. Thus, we report on the toxicity of E.BMP-2 in Sprague-Dawley rats under the condition of repetitive injection for 2 weeks. Randomly selected 10 male and female rats were administered with E.BMP-2 at a dose of 0.05, 0.18 or 0.5 mg/kg as an experimental group. A control group with another 10 rats was given E.BMP-2 carrier. Both E.BMP-2 and E.BMP-2 carrier were administered through intravenous injection for 2 weeks. For toxicokinetics study, 3 male and female rats were randomly selected from each group. During the observation period, general symptom, weight and food intakes were monitored, and ophthalmic and urine tests were performed as well. After the observation period, all animals were subjected to blood test, biochemical analysis and organ-weight measurement. During autopsy, visual inspections and histopathological examinations were done. Toxicokinetics study confirmed systemic exposure of the test material. No death or abnormal clinical sign was found during the injection period. Toxicity changes induced by the injection were not detected in autopsy or the tests for weight, food intakes, ophthalmology, hematology and serum biochemistry. The female groups administered with 0.18 and 0.5 mg/kg (the female 0.18-mg/kg group and the female 0.5-mg/kg group) showed absolute and relative weight loss in ovaries and reduced corpora lutea. It was the expected pharmacologic activity, rather than toxicity. The histopathological test revealed cartilage formation and increased fibroblast around the tail vein, but these were thought to be the result of osteoinductivity of the test material. In the male group with 0.5 mg/kg of E.BMP-2 (the male 0.5-mg/kg group), local appearance of multinucleated cells in lung parenchyma was observed, but it was considered as the natural reaction to remove E.BMP-2, which is a recombinant protein. In toxicokinetics study, systemic exposure (area under the serum concentration-time curve and maximum observed serum concentration) increased as the injection dose was increased in both male and female rats, and no clear difference was noticed between the sexes. Blood drug content did not change during the injection period, but the half-life was shortened as the injection dose was increased. Under the condition of this study, the no observed adverse effects level of E.BMP-2 was over 0.5 mg/kg in both male and female rats.

BMP-2 and bFGF release and in vitro effect on human osteoblasts after adsorption to bone grafts and biomaterials.

OBJECTIVES: Combination of scaffolds and growth factors is a promising option for several clinical problems in bone biomaterials. Simplified growth factor loading by adsorption from aqueous solution is one important option for this technology. We evaluated the adsorption followed by PBS rinsing, release and biological effect of transient loading with basic fibroblast growth factor (bFGF) and bone morphogenic protein 2 (BMP-2) on fresh frozen bone, processed bone matrix, collagen, and a ceramic material with immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and qRT-PCR. MATERIALS AND METHODS: The study consisted of three in vitro experiments (immunofluorescence, ELISA, and qRT-PCR) in human osteoblasts (HOB). The first evaluated the adsorption of the growth factors bFGF and BMP-2 to the biomaterials, analyzed by immunofluorescence assays. The second experiment used ELISA to analyze the release of the growth factors from the matrix. The biological effect of the growth factors on HOB was then studied with qRT-PCR experiments as the third step. RESULTS: Strongest sustained release peaks in ELISA were observed in bFGF loading on processed bone matrix (steam-resistant mineralized bone matrix, SMBM) with up to 553 pg/ml medium. BMP-2 loading was less effective in ELISA peak release experiments with up to 257 pg/ml medium in processed bone matrix (SMBM). bFGF showed also higher release peaks in collagen material (192 pg/ml) compared with BMP-2 (101 pg/ml). Cumulative release values 0-72 h were estimated. The expression of runX2, osteocalcin, and alkaline phosphatase as markers for osteoblast activity was correlating. CONCLUSION: The results showed sustained release of BMP-2 and bFGF after transient loading on bone biomaterials with a stronger effect in biological scaffolds. This is interesting for therapeutic growth factor loading as well as insights in natural growth factor matrix deposition during bone healing.

Evaluation of collagen/heparin coated TCP/HA granules for long-term delivery of BMP-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. The purpose of this study was to develop a sustained delivery system for recombinant human bone morphogenetic protein-2 (BMP-2). We covalently attached heparin to a cross-linked collagen type I coated tricalciumphosphate/hydroxyapatite (TCP/HA) bone substitute and subsequently loaded it with BMP-2. To systematically evaluate the contribution of each component with respect to the binding and release of BMP-2, six constructs were prepared and characterized: TCP/HA, TCP/HA with collagen (TCP/HACol), and TCP/HA with collagen and heparin (TCP/HAColHep) with and without BMP-2 (B). More BMP-2 bound to the TCP/HAColHep + B (92.9 ± 4.8 ng BMP-2/mg granule) granules as compared to the TCP/HACol + B (69.0 ± 9.6 ng BMP-2/mg granule) and TCP/HA + B granules (62.9 ± 5.4 ng BMP-2/mg granule). No difference in release pattern was found between the TCP/HA + B and TCP/HACol + B granules. Up to day 14, BMP-2 was still bound to the TCP/HAColHep + B granules, whereas most BMP had been released from TCP/HACol + B and TCP/HA + B granules at that time. After 21 days most BMP-2 also had been released from the TCP/HAColHep + B granules. The local and sustained delivery system for BMP-2 developed in this study may be useful as a carrier for BMP-2 and could possibly enhance bone regeneration efficacy for the treatment of large bone defects.

Morphological differences in BMP-2-induced ectopic bone between solid and crushed hyaluronan hydrogel templates.

The possibility to affect bone formation by using crushed versus solid hydrogels as carriers for bone morphogenetic protein 2 (BMP-2) was studied. Hydrogels, based on chemical crosslinking between hyaluronic acid and poly(vinyl alcohol) derivatives, were loaded with BMP-2 and hydroxyapatite. Crushed and solid forms of the gels were analyzed both in vitro via a release study using ¹²5I radioactive labeling of BMP-2, and in vivo in a subcutaneous ectopic bone model in rats. Dramatically different morphologies were observed for the ectopic bone formed in vivo in the two types of gels, even though virtually identical release profiles were observed in vitro. Solid hydrogels induced formation of a dense bone shell around non-degraded hydrogel, while crushed hydrogels demonstrated a uniform bone formation throughout the entire sample. These results suggest that by crushing the hydrogel, the construct's three-dimensional network becomes disrupted. This could expose unreacted functional groups, making the fragment's surfaces reactive and enable limited chemical fusion between the crushed hydrogel fragments, leading to similar in vitro release profiles. However, in vivo these interactions could be broken by enzymatic activity, creating a macroporous structure that allows easier cell infiltration, thus, facilitating bone formation.

Characterization of the chemically deposited hydroxyapatite coating on a titanium substrate.

Bioactive hydroxyapatite (HA) coating on titanium (Ti) implant can be used as a drug delivery device. A controlled release of drug around the implant requires the incorporation of drug into the coating material during the coating process. HA coating was prepared using a two-step procedure in conditions suitable for simultaneous incorporation of the protein-based drug into the coating material. Monetite coating was deposited on Ti substrate in acidic condition followed by the transformation of the monetite coating to HA. X-ray diffraction (XRD) confirmed the formation of the monetite phase at the first step of the coating preparation, which was transformed into HA at the second step. Fourier transform infrared spectroscopy demonstrated typical bands of a crystallized carbonated HA with A- and B-type substitution, which was confirmed by the XRD refinement of the structural parameters. Scanning electron microscope was used to observe the morphology of monetite and HA coatings. Adhesion of the coatings was measured using a scratch tester. The critical shearing stress was found to be 84.20 ± 1.27 MPa for the monetite coating, and 44.40 ± 2.39 MPa for the HA coating.

Assessing the character of the rhBMP-2- and vancomycin-loaded calcium sulphate composites in vitro and in vivo.

BACKGROUND: The treatment of contaminated and infected bone defects remains an intractable problem and the ideal approach is to control infection and repair the bone defect at the same time. Thus, developing an osteoconductive bone graft composite with antibiotic and growth factor release capabilities as well as osteogenesis-matched degradation properties is necessary. A new calcium sulphate composite consisting of vancomycin and rhBMP-2 was developed, and the present study assessed its efficiency in vitro and in a rabbit tibial defect model. METHODS: Firstly, we detected the bioactivity of rhBMP-2 released from the composites by ALP assay in vitro. Then, the released vancomycin in bone tissue within 1 cm from implanted site was detected by HLPC at 1, 3, 5, 7, 14, 21 and 28 days after implantation. The rhBMP-2 concentration of tissues around the defects was also detected by ELISA. Histomorphometry and histomorphometrical analysis at 5, 14 and 28 days post-implantation was done for assessing its osteoinductivity for bone defects. RESULTS: The results showed rhBMP-2 was still active in vitro at 29 days. In vivo, the composite released an initial bolus of vancomycin and rhBMP-2 to the bone followed by gradual release for more than 14 and 21 days, respectively. The histomorphometry indicated that the composite significantly augmented new bone formation in the defect compared to the control. CONCLUSIONS: This composite may be a potential therapeutic agent for contaminated or infected bone defects due to its concomitant osteoinductive and antibiotic properties.

Varying the sustained release of BMP-2 from chitosan nanogel-functionalized polycaprolactone fiber mats by different polycaprolactone surface modifications.

Polycaprolactone (PCL) fiber mats with different surface modifications were functionalized with a chitosan nanogel coating to attach the growth factor human bone morphogenetic protein 2 (BMP-2). Three different hydrophilic surface modifications were compared with regard to the binding and in vitro release of BMP-2. The type of surface modification and the specific surface area derived from the fiber thickness had an important influence on the degree of protein loading. Coating the PCL fibers with polydopamine resulted in the binding of the largest BMP-2 quantity per surface area. However, most of the binding was irreversible over the investigated period of time, causing a low release in vitro. PCL fiber mats with a chitosan-graft-PCL coating and an additional alginate layer, as well as PCL fiber mats with an air plasma surface modification boundless BMP-2, but the immobilized protein could almost completely be released. With polydopamine and plasma modifications as well as with unmodified PCL, high amounts of BMP-2 could also be attached directly to the surface. Integration of BMP-2 into the chitosan nanogel functionalization considerably increased binding on all hydrophilized surfaces and resulted in a sustained release with an initial burst release of BMP-2 without detectable loss of bioactivity in vitro.

Bone regeneration in ceramic scaffolds with variable concentrations of PDRN and rhBMP-2.

This study evaluated the bone regeneration capacity and mechanical properties of block-type hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds in response to different concentrations of polydeoxyribonucleotide (PDRN) and recombinant human bone morphogenic protein 2 (rhBMP-2). Thirty-two male white rabbits were used as a model of calvarial bone defect and classified into eight groups according to type and concentration of growth factor administered, viz., control group (only HA/TCP scaffold), scaffold + PDRN (0.1, 1, 5, and 10 mg/mL each) and scaffold + rhBMP-2 (0.01, 0.05, and 0.1 mg/mL each). The specimens were evaluated using histomorphometric and radiological analyses. Histomorphometric analyses indicated that the administration of PDRN did not increase bone formation. However, significant increases in bone formation were observed with the administration of rhBMP-2 at 0.05 and 0.10 mg/mL on week 8 compared to the control (p < 0.05). Radiological analyses revealed a significant increase in bone formation at week 8 with the administration of PDRN at 5 mg/mL and 10 mg/mL, and rhBMP-2 at 0.05 or 0.10 mg/mL compared to the control (p < 0.05). Our findings show that block-type HA/TCP scaffolds possess sufficient mechanical strength and bone regeneration capacity when used with optimal concentrations of growth factors.

A Chemotactic Functional Scaffold with VEGF-Releasing Peptide Amphiphiles Facilitates Bone Regeneration by BMP-2 in a Large-Scale Rodent Cranial Defect Model.

BACKGROUND: Current common techniques for repairing calvarial defects by autologous bone grafting and alloplastic implants have significant limitations. In this study, the authors investigated a novel alternative approach to bone repair based on peptide amphiphile nanofiber gels that are engineered to control the release of vascular endothelial growth factor (VEGF) to recruit circulating stem cells to a site of bone regeneration and facilitate bone healing by bone morphogenetic protein-2 (BMP-2). METHODS: VEGF release kinetics from peptide amphiphile gels were evaluated. Chemotactic functional scaffolds were fabricated by combining collagen sponges with peptide amphiphile gels containing VEGF. The in vitro and in vivo chemotactic activities of the scaffolds were evaluated by measuring mesenchymal stem cell migration, and angiogenic capability of the scaffolds was also evaluated. Large-scale rodent cranial bone defects were created to evaluate bone regeneration after implanting the scaffolds and other control materials. RESULTS: VEGF was released from peptide amphiphile in a controlled-release manner. In vitro migration of mesenchymal stem cells was significantly greater when exposed to chemotactic functional scaffolds compared to control scaffolds. In vivo chemotaxis was evidenced by migration of tracer-labeled mesenchymal stem cells to the chemotactic functional scaffolds. Chemotactic functional scaffolds showed significantly increased angiogenesis in vivo. Successful bone regeneration was noted in the defects treated with chemotactic functional scaffolds and BMP-2. CONCLUSIONS: The authors' observations suggest that this bioengineered construct successfully acts as a chemoattractant for circulating mesenchymal stem cells because of controlled release of VEGF from the peptide amphiphile gels. The chemotactic functional scaffolds may play a role in the future design of clinically relevant bone graft substitutes for large-scale bone defects.

A dose- and time-controllable syngeneic animal model of breast cancer microcalcification.

The development of novel diagnostic agents for the detection of breast cancer microcalcifications requires a reliable animal model. Based on previous work from our group, we hypothesized that a single systemic injection of recombinant bone morphogenetic protein-2 (rBMP-2) could be used to create such a model. The cDNA encoding mature human BMP-2 was expressed in BL21(DE3) bacteria, purified to homogeneity, and refolded as a dimer. Bioactivity was confirmed using a C2C12 alkaline phosphatase assay. rBMP-2 was radiolabeled with (99m)Tc, and its biodistribution and clearance were quantified after both intravenous (IV) and intraperitoneal (IP) injection. Fischer 344 rats bearing syngeneic R3230 breast tumors received a single intraperitoneal injection of rBMP-2 at a specified dose. Tumor microcalcification was quantified over time using micro-single photon emission computed tomography (SPECT) and microcomputed tomography (CT). rBMP-2 could be expressed in E. coli at high levels, isolated at >95% purity, and refolded to a bioactive dimer. Beta-phase half-life was 30.5 min after IV administration and 47.6 min after IP administration. Renal excretion was the primary mode of clearance. A single IP injection of >or=50 microg rBMP-2 when tumors were not yet palpable resulted in dose-dependent microcalcification in 8 of 8 R3230 tumors. No calcification was found in control tumors or in normal tissues and organs of animals injected with rBMP-2. Tumor calcification increased progressively between weeks 2 and 4 post-rBMP-2 injection. A single IP injection of rBMP-2 in rats bearing a syngeneic breast cancer will produce dose-dependent and time-dependent microcalcifications. This animal model lays the foundation for the development of novel diagnostic radiotracers for breast cancer.

Effect of scaffold architecture and BMP-2/BMP-7 delivery on in vitro bone regeneration.

The aim of this study was to develop 3-D tissue engineered constructs that mimic the in vivo conditions through a self-contained growth factor delivery system. A set of nanoparticles providing the release of BMP-2 initially followed by the release of BMP-7 were incorporated in poly(ε-caprolactone) scaffolds with different 3-D architectures produced by 3-D plotting and wet spinning. The release patterns were: each growth factor alone, simultaneous, and sequential. The orientation of the fibers did not have a significant effect on the kinetics of release of the model protein BSA; but affected proliferation of bone marrow mesenchymal stem cells. Cell proliferation on random scaffolds was significantly higher compared to the oriented ones. Delivery of BMP-2 alone suppressed MSC proliferation and increased the ALP activity to a higher level than that with BMP-7 delivery. Proliferation rate was suppressed the most by the sequential delivery of the two growth factors from the random scaffold on which the ALP activity was the highest. Results indicated the distinct effect of scaffold architecture and the mode of growth factor delivery on the proliferation and osteogenic differentiation of MSCs, enabling us to design multifunctional scaffolds capable of controlling bone healing.