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Impact Statement: Biomaterials can play a dual role in bone regeneration: they enable local sustained delivery of growth factors, such as bone morphogenetic protein-2 (BMP-2), while they provide structural support as scaffold. By better imitating the properties of native bone tissue, scaffolds may be both osteoconductive and osteoinductive. The latter can be achieved by modifying the electrical charge of the surface. The present work uses tunable oligo[(polyethylene glycol) fumarate] hydrogel and demonstrates that negative charge enhances BMP-2-induced bone formation compared with neutral or positive charge. Altogether, this indicates that tissue-specific surface charge modifications of biomaterials hold great promise in the field of tissue regeneration.

Purpose: Revascularization of natural and synthetic scaffolds is a critical part of the scaffold's incorporation and tissue ingrowth. Our goals were to create a biocompatible polymer scaffold with 3D-printing technology, capable of sustaining vascularization and tissue ingrowth., Methods: We synthesized biodegradable polycaprolactone fumarate (PCLF) scaffolds to allow tissue ingrowth via large interconnected pores. The scaffolds were prepared with Poly(lactic-co-glycolic acid)(PLGA) microspheres seeded with or without different growth factors including VEGF,FGF-2, and/or BMP-2. Scaffolds were implanted into the subcutaneous tissues of rats before undergoing histologic and microCT angiographic analysis., Results: At harvest after 12 weeks, scaffolds had tissue infiltrating into their pores without signs of scar tissue formation, fibrous capsule formation, or immune responses against PCLF. Histology for M1/M2 macrophage phenotypes confirmed that there were no overt signs of immune responses. Both microCT angiography and histologic analysis demonstrated marked tissue and vessel ingrowth throughout the pores traversing the body of the scaffolds. Scaffolds seeded with microspheres containing VEGF or VEGF with either BMP-2 or FGF-2 had significantly higher vascular ingrowth and vessel penetration than controls. All VEGF-augmented scaffolds were positive for Factor-VIII and exhibited collagen tissue infiltration throughout the pores. Furthermore, scaffolds with VEGF and BMP-2 had high levels of mineral deposition throughout the scaffold that are attributable to BMP-2., Conclusions: PCLF polymer scaffold can be utilized as a framework for vascular ingrowth and regeneration of multiple types of tissues. This novel scaffold material has promise in tissue regeneration across all types of tissues from soft tissue to bone.

Local sustained delivery of bioactive molecules from biomaterials is a promising strategy to enhance bone regeneration. To optimize delivery vehicles for bone formation, the design characteristics are tailored with consequential effect on bone morphogenetic protein-2 (BMP-2) release and bone regeneration. Complying with the 3R principles (Replacement, Reduction, and Refinement), the growth factor release is often investigated in vitro using several buffers to mimic the in vivo physiological environment. However, this remains an unmet need. Therefore, this study investigates the in vitro-in vivo correlation (IVIVC) of BMP-2 release from complex delivery vehicles in several commonly used in vitro buffers: cell culture model, phosphate buffered saline, and a strong desorption buffer. The results from this study showed that the release environment affected the BMP-2 release profiles, creating distinct relationships between release versus time and differences in extent of release. According to the guidance set by the U.S. Food and Drug Administration (FDA), IVIVC resulted in level A internal predictability for individual composites. Since the IVIVC was influenced by the BMP-2 loading method and composite surface chemistry, the external predictive value of the IVIVCs was limited. These results show that the IVIVCs can be used for predicting the release of an individual composite. However, the models cannot be used for predicting in vivo release for different composite formulations since they lack external predictability. Potential confounding effects of drug type, delivery vehicle formulations, and application site should be added to the equation to develop one single IVIVC applicable for complex delivery vehicles. Altogether, these results imply that more sophisticated in vitro systems should be used in bone regeneration to accurately discriminate and predict in vivo BMP-2 release from different complex delivery vehicles.

The optimal release profile of locally delivered bone morphogenetic protein-2 (BMP-2) for safe and effective clinical application is unknown. In this work, the effect of differential BMP-2 release on bone formation was investigated using a novel biomaterial oligo[(polyethylene glycol) fumarate] bis[2-(methacryloyloxy) ethyl] phosphate hydrogel (OPF-BP) containing poly(lactic-co-glycolic acid) microspheres. Three composite implants with the same biomaterial chemistry and structure but different BMP-loading methods were created: BMP-2 encapsulated in microspheres (OPF-BP-Msp), BMP-2 encapsulated in microspheres and adsorbed on the phosphorylated hydrogel (OPF-BP-Cmb), and BMP-2 adsorbed on the phosphorylated hydrogel (OPF-BP-Ads). These composites were compared with the clinically used BMP-2 carrier, Infuse® absorbable collagen sponge (ACS). Differential release profiles of bioactive BMP-2 were achieved by these composites. In a rat subcutaneous implantation model, OPF-BP-Ads and ACS generated a large BMP-2 burst release (>75%), whereas a more sustained release was seen for OPF-BP-Msp and OPF-BP-Cmb (~25% and 50% burst, respectively). OPF-BP-Ads generated significantly more bone than did all other composites, and the bone formation was 12-fold higher than that of the clinically used ACS. Overall, this study clearly shows that BMP-2 burst release generates more subcutaneous bone than do sustained release in OPF-BP-microsphere composites. Furthermore, composites should not only function as a delivery vehicle but also provide a proper framework to achieve appropriate bone formation., (Copyright © 2018 John Wiley & Sons, Ltd.)

Intra-articular ligamentous injuries are typically unrepairable and have limited outcomes after graft reconstruction. A combination of porous polycaprolactone fumarate (PCLF) scaffolds with polyethylene terephthalate (PET) sutures was developed with the goal of regenerating intra-articular ligaments. Scaffolds were fabricated by injecting PCLF over three-dimensional-printed molds containing two strands of PET suture down its central pore followed by cross-linking. Scaffolds were seeded with human mesenchymal stem cells (MSCs) from adipose tissue. To demonstrate cell attachment and proliferation in culture, we performed live/dead staining and cell proliferation assays. These experiments showed that MSCs remain viable and continue to proliferate on the scaffolds in culture for at least 2 weeks. Bare scaffolds were then used to reconstruct the rabbit anterior-cruciate ligament (ACL), while control rabbits underwent semitendinosus autograft reconstruction. The specimens underwent micro-computed tomography (CT) imaging, histological examination, and biomechanical testing at 8 weeks. The ultimate pull-out strength of the PCLF-PET scaffolds and tendon autografts was initially 72 ± 30 N and to 45 ± 10 N, respectively (p < 0.06). On inspection after 8 weeks in vivo, the intra-articular portion of the PCLF-PET scaffolds was fragmented while the tendon autografts remained intact. Cross-sectional areas of bone tunnels in the PCLF-PET scaffolds (11.3 ± 1 mm 2 ) were enlarged compared to tendon autografts (3.8 ± 0.5 mm 2 ) (p < 0.004) as measured by micro-CT. These studies show that PET-reinforced PCLF scaffolds are capable of initial ACL reconstruction and supports stem cell growth. The intra-articular portion of the scaffold may need to be re-engineered to support their use in ligament regeneration.

Positively-charged oligo[poly(ethylene glycol)fumarate] (OPF + ) is a biodegradable hydrogel used for spinal cord injury repair. We compared scaffolds containing primary Schwann cells (SCs) to scaffolds delivering SCs genetically modified to secrete high concentrations of glial cell-derived neurotrophic factor (GDNF). Multichannel OPF + scaffolds loaded with SCs or GDNF-SCs were implanted into transected rat spinal cords for 4 weeks. GDNF-SCs promoted regeneration of more axons into OPF + scaffolds (2773.0 ± 396.0) than primary SC OPF + scaffolds (1666.0 ± 352.2) (p = 0.0491). This increase was most significant in central and ventral-midline channels of the scaffold. Axonal remyelination was quantitated by stereologic analysis. Increased myelination of regenerating axons was observed in the GDNF-SC group. Myelinating cell and axon complexes were formed by host SCs and not by implanted cells or host oligodendrocytes. Fast Blue retrograde tracing studies determined the rostral-caudal directionality of axonal growth. The number of neurons that projected axons rostrally through the GDNF-SC scaffolds was higher (7929 ± 1670) than in animals with SC OPF + scaffolds (1069 ± 241.5) (p < 0.0001). The majority of ascending axons were derived from neurons located more than 15 mm from the scaffold-cord interface, and were identified to be lumbosacral intraspinal motor neurons. Transected animals with GDNF-SC OPF + scaffolds partially recovered locomotor function at weeks 3 and 4 following surgery. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Current treatment options for cartilage injuries are limited. The goals of this study are to create a biodegradable polymer scaffold with the capabilities of sustaining chondrocyte growth and proliferation, enable cell-to-cell communication and tissue regeneration through large pores, and assess the biological augmentation of the scaffold capabilities using platelet lysate (PL). We synthesized biodegradable polycaprolactone fumarate (PCLF) scaffolds to allow cell-cell communication through large interconnected pores. Molds were printed using a three-dimensional printer and scaffolds synthesized through UV crosslinking. Culture medium included alpha modified Eagle's media with either 10% fetal bovine serum (FBS) or 5% PL, a mixture of platelet release products, after being seeded onto scaffolds through a dynamic bioreactor. Assays included cellular proliferation (MTS), toxicity and viability (live/dead immunostaining), differentiation (glycosaminoglycan [GAG], alkaline phosphatase [ALP], and total collagen), and immunostaining for chondrogenic markers collagen II and Sox 9 (with collagen I as a negative control). The large interconnected pores (500 and 750 μm) enable cell-to-cell communication and cellular infiltration into the scaffolds, as the cells remained viable and proliferated for 2 weeks. Chondrocytes cultured in PL showed increased rates of proliferation when compared with FBS. The chondrogenic markers GAG and total collagen contents increased over 2 weeks at each time point, whereas the osteogenic marker ALP did not significantly change. Immunostaining at 2 and 4 weeks for the expression of chondrogenic markers Collagen II and Sox 9 was increased when compared with control human fibroblasts. These results show that the PCLF polymer scaffold enables chondrocytes to attach, proliferate, and retain their chondrogenic phenotypes, demonstrating potential in chondrocyte engineering and cartilage regeneration.

Anterior cruciate ligament (ACL) ruptures reconstructed with tendon grafts are commonly fixed with bioabsorbable implants, which are frequently complicated by incomplete bone filling upon degradation. Bone regeneration after ACL reconstruction could be enhanced by utilizing tissue engineering techniques and three-dimensional (3D) printing to create a porous bioabsorbable scaffold with delayed delivery of recombinant-human bone morphogenetic protein 2 (rhBMP-2). The first aim of this study was to design a 3D poly(propylene fumarate) (PPF) porous scaffold that maintained suitable pullout strength for future testing in a rabbit ACL reconstruction model. Our second aim was to determine the release kinetics of rhBMP-2 from PPF scaffolds that utilized both calcium-phosphate coatings and growth factor delivery on microspheres, both of which have been shown to decrease the initial burst release of rhBMP-2 and increase bone regeneration. To determine the degree of scaffold porosity that maintained suitable pullout strength, tapered scaffolds were fabricated with increasing porosity (0%, 20%, 35%, and 44%) and pullout testing was performed in a cadaveric rabbit ACL reconstruction model. Scaffolds were coated with carbonate hydroxyapatite (synthetic bone mineral [SBM]), and radiolabeled rhBMP-2 was delivered in four different experimental groups as follows: Poly(lactic-co-glycolic acid) microspheres only, microspheres and collagen (50:50), collagen only, and saline solution only. rhBMP-2 release was measured at day 1, 2, 4, 8, 16, and 32. The microsphere delivery groups had a smaller burst release and released a smaller percentage of rhBMP-2 over the 32 days than the collagen and saline only groups. In conclusion, a porous bioabsorbable scaffold with suitable strength for a rabbit ACL reconstruction was developed. Combining a synthetic bone mineral coating with microspheres had an additive effect, decreasing the initial burst release and cumulative release of rhBMP-2. Future studies need to evaluate this scaffold's fixation strength and bone filling capabilities in vivo compared to traditional bioabsorbable implants.

Surgical site infection (SSI) remains a significant risk for any clean orthopedic surgical procedure. Complications resulting from an SSI often require a second surgery and lengthen patient recovery time. The efficacy of antimicrobial agents delivered to combat SSI is diminished by systemic toxicity, bacterial resistance, and patient compliance to dosing schedules. We submit that development of localized, controlled release formulations for antimicrobial compounds would improve the effectiveness of prophylactic surgical wound antibiotic treatment while decreasing systemic side effects. Our research group developed and characterized oligo(poly(ethylene glycol)fumarate)/sodium methacrylate (OPF/SMA) charged copolymers as biocompatible hydrogel matrices. Here, we report the engineering of this copolymer for use as an antibiotic delivery vehicle in surgical applications. We demonstrate that these hydrogels can be efficiently loaded with vancomycin (over 500 μg drug per mg hydrogel) and this loading mechanism is both time- and charge-dependent. Vancomycin release kinetics are shown to be dependent on copolymer negative charge. In the first 6 hours, we achieved as low as 33.7% release. In the first 24 hours, under 80% of total loaded drug was released. Further, vancomycin release from this system can be extended past four days. Finally, we show that the antimicrobial activity of released vancomycin is equivalent to stock vancomycin in inhibiting the growth of colonies of a clinically derived strain of methicillin-resistant Staphylococcus aureus. In summary, our work demonstrates that OPF/SMA hydrogels are appropriate candidates to deliver local antibiotic therapy for prophylaxis of surgical site infection.

Purpose: Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer "neoligament" seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue., Methods: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell-cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C)., Results: AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p < 0.05). Cells had a low baseline expression of ALP and GAG, but increased expression of total collagen when induced by the ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p < 0.01) instead of FBS (p < 0.05). FGF-2 and PL also significantly increased immunostaining of tenascin-C and collagen at 2 and 4 weeks compared with human fibroblasts., Summary: Our results demonstrate that AMSCs proliferate and eventually produce a collagen-rich extracellular matrix on porous PCLF scaffolds. This novel scaffold has potential in stem cell engineering and ligament regeneration.

In tissue engineering, development of an osteoconductive construct that integrates with host tissue remains a challenge. In this work, the effect of bone-like minerals on maturation of pre-osteoblast cells was investigated using polymer-mineral scaffolds composed of poly(propylene fumarate)-co-poly(caprolactone) (PPF-co-PCL) and nano-sized hydroxyapatite (HA). The HA of varying concentrations was added to an injectable formulation of PPF-co-PCL and the change in thermal and mechanical properties of the scaffolds was evaluated. No change in onset of degradation temperature was observed due to the addition of HA, however compressive and tensile moduli of copolymer changed significantly when HA amounts were increased in composite formulation. The change in mechanical properties of copolymer was found to correlate well to HA concentration in the constructs. Electron microscopy revealed mineral nucleation and a change in surface morphology and the presence of calcium and phosphate on surfaces was confirmed using energy dispersive X-ray analysis. To characterize the effect of mineral on attachment and maturation of pre-osteoblasts, W20-17 cells were seeded on HA/copolymer composites. We demonstrated that cells attached more to the surface of HA containing copolymers and their proliferation rate was significantly increased. Thus, these findings suggest that HA/PPF-co-PCL composite scaffolds are capable of inducing maturation of pre-osteoblasts and have the potential for use as scaffold in bone tissue engineering., (© 2014 Wiley Periodicals, Inc.)

Positively charged oligo[poly(ethylene glycol) fumarate] (OPF+) scaffolds loaded with Schwann cells bridge spinal cord injury (SCI) lesions and support axonal regeneration in rat. The regeneration achieved is not sufficient for inducing functional recovery. Attempts to increase regeneration would benefit from understanding the effects of the scaffold and transplanted cells on lesion environment. We conducted morphometric and stereological analysis of lesions in rats implanted with OPF+ scaffolds with or without loaded Schwann cells 1, 2, 3, 4, and 8 weeks after thoracic spinal cord transection. No differences were found in collagen scarring, cyst formation, astrocyte reactivity, myelin debris, or chondroitin sulfate proteoglycan (CSPG) accumulation. However, when scaffold-implanted animals were compared with animals with transection injuries only, these barriers to regeneration were significantly reduced, accompanied by increased activated macrophages/microglia. This distinctive and regeneration permissive tissue reaction to scaffold implantation was independent of Schwann cell transplantation. Although the tissue reaction was beneficial in the short term, we observed a chronic fibrotic host response, resulting in scaffolds surrounded by collagen at 8 weeks. This study demonstrates that an appropriate biomaterial scaffold improves the environment for regeneration. Future targeting of the host fibrotic response may allow increased axonal regeneration and functional recovery.

Various calcium phosphate based coatings have been evaluated for better bony integration of metallic implants and are currently being investigated to improve the surface bioactivity of polymeric scaffolds. The aim of this study was to evaluate the role of calcium phosphate coating and simultaneous delivery of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the in vivo bone regeneration capacity of biodegradable, porous poly(propylene fumarate) (PPF) scaffolds. PPF scaffolds were coated with three different calcium phosphate formulations: magnesium-substituted β-tricalcium phosphate (β-TCMP), carbonated hydroxyapatite (synthetic bone mineral, SBM) and biphasic calcium phosphate (BCP). In vivo bone regeneration was evaluated by implantation of scaffolds in a critical-sized rabbit calvarial defect loaded with different doses of rhBMP-2. Our data demonstrated that scaffolds with each of the calcium phosphate coatings were capable of sustaining rhBMP-2 release and retained an open porous structure. After 6weeks of implantation, micro-computed tomography revealed that the rhBMP-2 dose had a significant effect on bone formation within the scaffolds and that the SBM-coated scaffolds regenerated significantly greater bone than BCP-coated scaffolds. Mechanical testing of the defects also indicated restoration of strength in the SBM and β-TCMP with rhBMP-2 delivery. Histology results demonstrated bone growth immediately adjacent to the scaffold surface, indicating good osteointegration and osteoconductivity for coated scaffolds. The results obtained in this study suggest that the coated scaffold platform demonstrated a synergistic effect between calcium phosphate coatings and rhBMP-2 delivery and may provide a promising platform for the functional restoration of large bone defects., (Copyright © 2015. Published by Elsevier Ltd.)

Aim: Critical knee osteochondral defects in seven adult minipigs were treated with oligo(polyethylene glycol)fumarate (OPF) hydrogel combined with autologous or human adipose-derived stem cells (ASCs), and evaluated after 6 months., Methods: Four defects were made on the peripheral part of right trochleas (n = 28), and treated with OPF scaffold alone or pre-seeded with ASCs., Results: A better quality cartilage tissue characterized by improved biomechanical properties and higher collagen type II expression was observed in the defects treated by autologous or human ASC-loaded OPF; similarly this approach induced the regeneration of more mature bone with upregulation of collagen type I expression., Conclusion: This study provides the evidence that both porcine and human adipose-derived stem cells associated to OPF hydrogel allow improving osteochondral defect regeneration in a minipig model.

We previously demonstrated recipient-derived neoangiogenesis to maintain viability of living bone allogeneic transplants without long-term immunosuppression. The effect of cytokine delivery to enhance this process is studied. Vascularized femur transplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(d,l-lactide-co-glycolide) microspheres loaded with buffer (N = 11), basic fibroblast growth factor (FGF2) (N = 10), vascular endothelial growth factor (VEGF) (N = 11), or both (N = 11) were inserted intramedullarly alongside a recipient-derived arteriovenous bundle. FK-506 was administered for 2 weeks. At 18 weeks, bone blood flow, microangiography, histologic, histomorphometric, and alkaline phosphatase measurements were performed. Bone blood flow was greater in the combined group than control and VEGF groups (P = 0.04). Capillary density was greater in the FGF2 group than in the VEGF and combined groups (P < 0.05). Bone viability, growth, and alkaline phosphatase activity did not vary significantly between groups. Neoangiogenesis in vascularized bone allotransplants is enhanced by angiogenic cytokine delivery, with results using FGF2 that are comparable to isotransplant from previous studies. Further studies are needed to achieve bone formation similar to isotransplants., (Copyright © 2013 Wiley Periodicals, Inc.)

A novel biodegradable copolymer, poly(propylene fumarate-co-caprolactone) [P(PF-co-CL)], has been developed in our laboratory as an injectable scaffold for bone defect repair. In the current study, we evaluated the ability of P(PF-co-CL) to reconstitute the load-bearing capacity of vertebral bodies with lytic lesions. Forty vertebral bodies from four fresh-frozen cadaveric thoracolumbar spines were used for this study. They were randomly divided into four groups: intact vertebral body (intact control), simulated defect without treatment (negative control), defect treated with P(PF-co-CL) (copolymer group), and defect treated with poly(methyl methacrylate) (PMMA group). Simulated metastatic lytic defects were made by removing a central core of the trabecular bone in each vertebral body with an approximate volume of 25% through an access hole in the side of the vertebrae. Defects were then filled by injecting either P(PF-co-CL) or PMMA in situ crosslinkable formulations. After the spines were imaged with quantitative computerized tomography, single vertebral body segments were harvested for mechanical testing. Specimens were compressed until failure or to 25% reduction in body height and ultimate strength and elastic modulus of each specimen were then calculated from the force-displacement data. The average failure strength of the copolymer group was 1.83 times stronger than the untreated negative group and it closely matched the intact vertebral bodies (intact control). The PMMA-treated vertebrae, however, had a failure strength 1.64 times larger compared with the intact control. The elastic modulus followed the same trend. This modulus mismatch between PMMA-treated vertebrae and the host vertebrae could potentially induce a fracture cascade and degenerative changes in adjacent intervertebral discs. In contrast, P(PF-co-CL) restored the mechanical properties of the treated segments similar to the normal, intact, vertebrae. Therefore, P(PF-co-CL) may be a suitable alternative to PMMA for vertebroplasty treatment of vertebral bodies with lytic defects.

Four biomaterial tubes, poly(lactic-co-glycolic acid) (PLGA), poly(caprolactone fumarate) (PCLF), a neutral oligo[(polyethylene glycol) fumarate] (OPF) hydrogel or a positively charged oligo[(polyethylene glycol) fumarate] (OPF(+)) hydrogel with a PCLF sleeve, have previously been shown to have benefits for nerve repair. However, no direct comparison to identify the optimal material have been made. Herein, these nerve tubes were implanted in a rat sciatic nerve model and nerve regeneration was quantified and compared by using accepted nerve assessment techniques. Using standard statistical methods, no significant differences of individual parameters were apparent between groups despite PCLF showing a tendency to perform better than the others. Using a mean-variance based ranking system of multiple independent parameters, statistical differences became apparent. It was clear that the PLCF tube supported significantly improved nerve regeneration and recovery compared to the other three biomaterial conduits. The ability to simultaneously compare a number of regenerative parameters and elucidate the best material from the combination of these individual parameters is of importance to the nerve regeneration area and has implications for the tissue engineering field. By using this method of comparison, a number of biomaterial constructs may be compared under similar conditions and the optimal construct elucidated using the minimal number of animals and materials., (© 2013 Elsevier Ltd. All rights reserved.)

Osteosarcoma is a bone tumor that affects children and young adults. 2-Methoxyestradiol (2-ME), a naturally occurring estrogen metabolite, kills osteosarcoma cells, but does not affect normal osteoblasts. In order to effectively target osteosarcoma and improve the therapeutic index of the drug 2-ME, we have encapsulated 2-ME in a composite of oligo-(polyethylene glycol) fumarate (OPF) hydrogel and poly (lactic-co-glycolic acid) (PLGA) microspheres and investigated the effect of polymer composition on 2-ME release kinetics and osteosarcoma cell survival. The in vitro study shows that 2-ME can be released in a controlled manner over 21-days. The initial burst releases observed on day 1 were 50% and 32% for OPF and OPF/PLGA composites, respectively. The extended release kinetics show that 100% of the encapsulated 2-ME is released by day 12 from OPF, whereas the OPF/PLGA composites showed a release of 85% on day 21. 2-ME released from the polymers was biologically active and blocked osteosarcoma cell proliferation in vitro. Also, comparison of 2-ME delivery in osteosarcoma cells in culture, shows that direct treatment has no effect after 3 days, whereas polymer-mediated delivery produces anti-tumor effects that could be sustained for 21 days. These findings show that the OPF and PLGA polymeric system may prove to be useful in controlled and sustained delivery of 2-ME and could be further explored in the treatment of osteosarcoma., (Copyright © 2012 Wiley Periodicals, Inc.)

Treatment of large segmental bone defects remains an unsolved clinical challenge, despite a wide array of existing bone graft materials. This project was designed to rapidly assess and compare promising biodegradable osteoconductive scaffolds for use in the systematic development of new bone regeneration methodologies that combine scaffolds, sources of osteogenic cells, and bioactive scaffold modifications. Promising biomaterials and scaffold fabrication methods were identified in laboratories at Rutgers, MIT, Integra Life Sciences, and Mayo Clinic. Scaffolds were fabricated from various materials, including poly(L-lactide-co-glycolide) (PLGA), poly(L-lactide-co-ɛ-caprolactone) (PLCL), tyrosine-derived polycarbonate (TyrPC), and poly(propylene fumarate) (PPF). Highly porous three-dimensional (3D) scaffolds were fabricated by 3D printing, laser stereolithography, or solvent casting followed by porogen leaching. The canine femoral multi-defect model was used to systematically compare scaffold performance and enable selection of the most promising substrate(s) on which to add cell sourcing options and bioactive surface modifications. Mineralized cancellous allograft (MCA) was used to provide a comparative reference to the current clinical standard for osteoconductive scaffolds. Percent bone volume within the defect was assessed 4 weeks after implantation using both MicroCT and limited histomorphometry. Bone formed at the periphery of all scaffolds with varying levels of radial ingrowth. MCA produced a rapid and advanced stage of bone formation and remodeling throughout the defect in 4 weeks, greatly exceeding the performance of all polymer scaffolds. Two scaffold constructs, TyrPC(PL)/TCP and PPF4(SLA)/HA(PLGA) (Dip), proved to be significantly better than alternative PLGA and PLCL scaffolds, justifying further development. MCA remains the current standard for osteoconductive scaffolds.

Stimuli-responsive hydrogels have enormous potential in drug delivery applications. They can be used for site-specific drug delivery due to environmental variables in the body such as pH and temperature. In this study, we have developed pH-responsive microgels for the delivery of doxorubicin (DOX) in order to optimize its anti-tumor activity while minimizing its systemic toxicity. We used a copolymer of oligo(polyethylene glycol) fumarate (OPF) and sodium methacrylate (SMA) to fabricate the pH-responsive microgels. We demonstrated that the microgels were negatively charged, and the amounts of charge on the microgels were correlated with the SMA concentration in their formulation. The resulting microgels exhibited sensitivity to the pH and ionic strength of the surrounding environment. We demonstrated that DOX was efficiently loaded into the microgels and released in a controlled fashion via an ion-exchange mechanism. Our data revealed that the DOX release was influenced by the pH and ionic strength of the solution. Moreover, we designed a phenomenological mathematical model, based on a stretched exponential function, to quantitatively analyze the cumulative release of DOX. We found a linear correlation between the maximum release of DOX calculated from the model and the SMA concentration in the microgel formulation. The anti-tumor activity of the released DOX was assessed using a human chordoma cell line. Our data revealed that OPF-SMA microgels prolonged the cell killing effect of DOX., (Copyright © 2012. Published by Elsevier Ltd.)

Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKO(OCN) mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKO(OCN) mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKO(OCN) mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKO(OCN) mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Poly(ε-caprolactone fumarate) (PCLF) scaffold formulations were assessed as a delivery system for 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. The data suggested that PCLF incorporated with PVA and HA accelerated rhBMP-2 release and that the released protein was bioactive. For the in vivo study, a critical-sized defect (CSD) model in 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 that PCLF delivered biologically active rhBMP-2, promoted bone healing in a CSD and has potential as a bone tissue engineering scaffold., (Copyright © 2011 John Wiley & Sons, Ltd.)

In this work, we have investigated the development of a synthetic hydrogel that contains a negatively charged phosphate group for use as a substrate for bone cell attachment and differentiation in culture. The photoreactive, phosphate-containing molecule, bis(2-(methacryloyloxy)ethyl)phosphate (BP), was incorporated into oligo(polyethylene glycol) fumarate hydrogel and the mechanical, rheological and thermal properties of the resulting hydrogels were characterized. Our results showed changes in hydrogel compression and storage moduli with incorporation of BP. The modification also resulted in decreased crystallinity as recorded by differential scanning calorimetry. Our data revealed that incorporation of BP improved attachment and differentiation of human fetal osteoblast (hFOB) cells in a dose-dependent manner. A change in surface chemistry and mineralization of the phosphate-containing surfaces verified by scanning electron microscopy and energy dispersive X-ray analysis was found to be important for hFOB cell attachment and differentiation. We also demonstrated that phosphate-containing hydrogels support attachment and differentiation of primary bone marrow stromal cells. These findings suggest that BP-modified hydrogels are capable of sustaining attachment and differentiation of both bone marrow stromal cells and osteoblasts that are critical for bone regeneration., (Copyright © 2012. Published by Elsevier Ltd.)

Vascular endothelial growth factor (VEGF) is a potent angiogenic stimulator. Controlled release of such stimulators may enhance and guide the vascularization process, and when applied in a nerve conduit may play a role in nerve regeneration. We report the fabrication and in vitro characterization of poly-lactic-co-glycolic acid (PLGA) microspheres encapsulating VEGF and the in vivo application of nerve conduits supplemented with VEGF-containing microspheres. PLGA microspheres containing VEGF were prepared by the double emulsion-solvent evaporation technique. This yielded 83.16% of microspheres with a diameter <53 μm. VEGF content measured by ELISA indicated 93.79±10.64% encapsulation efficiency. Release kinetics were characterized by an initial burst release of 67.6±8.25% within the first 24h, followed by consistent release of approximately 0.34% per day for 4 weeks. Bioactivity of the released VEGF was tested by human umbilical vein endothelial cell (HUVEC) proliferation assay. VEGF released at all time points enhanced HUVEC proliferation, confirming that VEGF retained its bioactivity throughout the 4 week time period. When the microsphere delivery system was placed in a biosynthetic nerve scaffold robust nerve regeneration was observed. This study established a novel system for controlled release of growth factors and enables in vivo studies of nerve conduits conditioned with this system., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The transected rat thoracic (T(9/10)) spinal cord model is a platform for quantitatively comparing biodegradable polymer scaffolds. Schwann cell-loaded scaffolds constructed from poly (lactic co-glycolic acid) (PLGA), poly(ɛ-caprolactone fumarate) (PCLF), oligo(polyethylene glycol) fumarate (OPF) hydrogel or positively charged OPF (OPF+) hydrogel were implanted into the model. We demonstrated that the mechanical properties (3-point bending and stiffness) of OPF and OPF + hydrogels closely resembled rat spinal cord. After one month, tissues were harvested and analyzed by morphometry of neurofilament-stained sections at rostral, midlevel, and caudal scaffold. All polymers supported axonal growth. Significantly higher numbers of axons were found in PCLF (P < 0.01) and OPF+ (P < 0.05) groups, compared to that of the PLGA group. OPF + polymers showed more centrally distributed axonal regeneration within the channels while other polymers (PLGA, PCLF and OPF) tended to show more evenly dispersed axons within the channels. The centralized distribution was associated with significantly more axons regenerating (P < 0.05). Volume of scar and cyst rostral and caudal to the implanted scaffold was measured and compared. There were significantly smaller cyst volumes in PLGA compared to PCLF groups. The model provides a quantitative basis for assessing individual and combined tissue engineering strategies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this study we have compared the effects of negative and positive fixed charges on chondrocyte behavior in vitro. Electrical charges have been incorporated into oligo(poly(ethylene glycol) fumarate) (OPF) using small charged monomers such as sodium methacrylate (SMA) and (2-(methacryloyloxy) ethyl)-trimethyl ammonium chloride (MAETAC) to produce negatively and positively charged hydrogels, respectively. The physical and electrical properties of the hydrogels were characterized by measuring and calculating the swelling ratio and zeta potential, respectively. Our results revealed that the properties of these OPF modified hydrogels varied according to the concentration of charged monomers. Zeta potential measurements demonstrated that the electrical properties of the OPF hydrogel surfaces changed on incorporation of SMA and MAETAC and that these changes in electrical properties were dose-dependent. Attenuated total reflectance Fourier transform infrared spectroscopy was used to determine the hydrogel surface composition. To assess the effects of surface properties on chondrocyte behavior primary chondrocytes isolated from rabbit ears were seeded as a monolayer on top of the hydrogels. We demonstrated that the cells remained viable over 7 days and began to proliferate while seeded on top of the hydrogels. Collagen type II staining was positive in all samples, however, the staining intensity was higher on negatively charged hydrogels. Similarly, glycosaminoglycan production was significantly higher on negatively charged hydrogels compared with a neutral hydrogel. Reverse transcriptase polymerase chain reaction showed up-regulation of collagen type II and down-regulation of collagen type I on the negatively charged hydrogels. These findings indicate that charge plays an important role in establishing an appropriate environment for chondrocytes and, hence, in the engineering of cartilage. Thus, further investigations into charged hydrogels for cartilage tissue engineering is merited., (Copyright © 2011. Published by Elsevier Ltd.)

This study describes the use of oligo [(polyethylene glycol) fumarate] (OPF) hydrogel scaffolds as vehicles for sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the transected spinal cord. dbcAMP was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within the scaffolds architecture. Functionality of the released dbcAMP was assessed using neurite outgrowth assays in PC12 cells and by delivery to the transected spinal cord within OPF seven channel scaffolds, which had been loaded with Schwann cells or mesenchymal stem cells (MSCs). Our results showed that encapsulation of dbcAMP in microspheres lead to prolonged release and continued functionality in vitro. These microspheres were then successfully incorporated into OPF scaffolds and implanted in the transected thoracic spinal cord. Sustained delivery of dbcAMP inhibited axonal regeneration in the presence of Schwann cells but rescued MSC-induced inhibition of axonal regeneration. dbcAMP was also shown to reduce capillary formation in the presence of MSCs, which was coupled with significant functional improvements. Our findings demonstrate the feasibility of incorporating PLGA microsphere technology for spinal cord transection studies. It represents a novel sustained delivery mechanism within the transected spinal cord and provides a platform for potential delivery of other therapeutic agents.

Polypropylene fumarate (PPF) scaffolds fabricated by rapid prototyping were surface modified by solution deposition of electrically conductive polypyrrole coatings with or without hydroxyapatite. Scaffolds were electrically conductive with resistivity as low as 2Ω. Scaffold characterization by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and thermo gravimetric analysis shows both polypyrrole and hydroxyapatite are present. Cell viability, attachment, proliferation, and differentiation were analyzed using human fetal osteoblast cells. These studies show that surface modification using hydroxyapatite improved cell attachment and proliferation of osteoblasts onto the PPF scaffolds. Alkaline phosphatase activity as a marker for osteogenic differentiation of cell to mature osteoblasts was analyzed. Our data reveal that osteoblasts maintained their phenotype on PPF scaffolds with and without coatings. Thus, these scaffolds could be appropriate candidates for our future in vivo studies.

In this work, a series of co-polymers of polypropylene fumarate-co-polycaprolactone (PPF-co-PCL) were synthesized via a three-step polycondensation reaction of oligomeric polypropylene fumarate (PPF) with polycaprolactone (PCL). The effects of PPF precursor molecular weight, PCL precursor molecular weight and PCL fraction in the co-polymer (PCL feed ratio) on the maximum cross-linking temperature, gelation time and mechanical properties of the cross-linked co-polymers were investigated. The maximum cross-linking temperature fell between 38.2 ± 0.3 and 47.2 ± 0.4°C, which increased with increasing PCL precursor molecular weight. The gelation time was between 4.2 ± 0.2 and 8.5 ± 0.7 min, and decreased with increasing PCL precursor molecular weight. The compressive moduli ranged from 44 ± 1.8 to 142 ± 7.4 MPa, with enhanced moduli at higher PPF precursor molecular weight and lower PCL feed ratio. The compressive toughness was in the range of 4.1 ± 0.3 and 17.1 ± 1.3 kJ/m(3). Our data suggest that the cross-linking and mechanical properties of PPF-co-PCL can be modulated by varying the composition. Therefore, the PPF-co-PCL co-polymers may offer increased versatility as an injectable, in situ polymerizable biomaterial than the individual polymers of PPF and PCL.

Electrically conductive hydrogel composites consisting of oligo(polyethylene glycol) fumarate (OPF) and polypyrrole (PPy) were developed for applications in nerve regeneration. OPF-PPy scaffolds were synthesized using three different anions: naphthalene-2-sulfonic acid sodium salt (NSA), dodecylbenzenesulfonic acid sodium salt (DBSA), and dioctyl sulfosuccinate sodium salt (DOSS). Scaffolds were characterized by ATR-FTIR, XPS, AFM, dynamic mechanical analysis, electrical resistivity measurements, and swelling experiments. OPF-PPy scaffolds were shown to consist of up to 25 mol % polypyrrole with a compressive modulus ranging from 265 to 323 kPa and a sheet resistance ranging from 6 to 30 × 10(3) Ohms/square. In vitro studies using PC12 cells showed OPF-PPy materials had no cytotoxicity and PC12 cells showed distinctly better cell attachment and an increase in the percent of neurite bearing cells on OPF-PPy materials compared to OPF. The neurite lengths of PC12 cells were significantly higher on OPF-PPyNSA and OPF-PPyDBSA. These results show that electrically conductive OPF-PPy hydrogels are promising candidates for future applications in nerve regeneration.

The goal of this study was to develop a polymeric carrier for delivery of anti-tumor drugs and sustained release of these agents in order to optimize anti-tumor activity while minimizing systemic effects. We used oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels modified with small negatively charged molecules, sodium methacrylate (SMA), for delivery of doxorubicin (DOX). SMA at different concentrations was incorporated into the OPF hydrogel with a photo-crosslinking method. The resulting hydrogels exhibited sensitivity to the pH and ionic strength of the surrounding environment. Our results revealed that DOX was bound to the negatively charged hydrogel through electrostatic interaction and was released in a timely fashion with an ion-exchange mechanism. Release kinetics of DOX was directly correlated to the concentration of SMA in the hydrogel formulations. Anti-tumor activity of the released DOX was assessed using a human osteosarcoma cell line. Our data revealed that DOX released from the modified, charged hydrogels remained biologically active and had the capability to kill cancer cells. In contrast, control groups of unmodified OPF hydrogels with or without DOX did not exhibit any cytotoxicity. This study demonstrates the feasibility of using SMA-modified OPF hydrogels as a potential carrier for chemotherapeutic drugs for cancer treatments., (Copyright 2010 Elsevier Ltd. All rights reserved.)