Bone Tissue Engineering with Multilayered Scaffolds-Part II: Combining Vascularization with Bone Formation in Critical-Sized Bone Defect.

Our previous in vivo study showed that multilayered scaffolds made of an angiogenic layer embedded between an osteogenic layer and an osteoconductive layer, with layer thickness in the 100-400 µm range, resulted in through-the-thickness vascularization of the construct even in the absence of exogenous endothelial cells. The angiogenic layer was a collagen-fibronectin gel, and the osteogenic layer was made from nanofibrous polycaprolactone while the osteoconductive layer was made either from microporous hydroxyapatite or microfibrous polycaprolactone. In this follow-up study, we implanted these acellular and cellular multilayered constructs in critical-sized rat calvarial defects and evaluated their vascularization and bone formation potential. Vascularization and bone formation at the defect were evaluated and quantified using microcomputed tomography (microCT) followed by perfusion of the animals with the radio opaque contrast agent, MICROFIL. The extent of bony bridging and union within the critical-sized defect was evaluated using a previously established scoring system from the microCT data set. Similarly the new bone formation in the defect was quantified from the microCT data set as previously reported. Histological evaluation at 4 and 12 weeks validated the microCT findings. Our experimental results showed that acellular multilayered scaffolds with microscale-thick nanofibers and porous ceramic discs with angiogenic zone at their interface can regenerate functional vasculature and bone similar to that of cellular constructs in critical-sized calvarial defects. This result suggests that suitably bioengineered acellular multilayered constructs can be an improved and more translational approach in functional in vivo bone regeneration.

Autologously generated tissue-engineered bone flaps for reconstruction of large mandibular defects in an ovine model.

The reconstruction of large craniofacial defects remains a significant clinical challenge. The complex geometry of facial bone and the lack of suitable donor tissue often hinders successful repair. One strategy to address both of these difficulties is the development of an in vivo bioreactor, where a tissue flap of suitable geometry can be orthotopically grown within the same patient requiring reconstruction. Our group has previously designed such an approach using tissue chambers filled with morcellized bone autograft as a scaffold to autologously generate tissue with a predefined geometry. However, this approach still required donor tissue for filling the tissue chamber. With the recent advances in biodegradable synthetic bone graft materials, it may be possible to minimize this donor tissue by replacing it with synthetic ceramic particles. In addition, these flaps have not previously been transferred to a mandibular defect. In this study, we demonstrate the feasibility of transferring an autologously generated tissue-engineered vascularized bone flap to a mandibular defect in an ovine model, using either morcellized autograft or synthetic bone graft as scaffold material.

In vitro and in vivo evaluation of self-mineralization and biocompatibility of injectable, dual-gelling hydrogels for bone tissue engineering.

In this study, we investigated the mineralization capacity and biocompatibility of injectable, dual-gelling hydrogels in a rat cranial defect as a function of hydrogel hydrophobicity from either the copolymerization of a hydrolyzable lactone ring or the hydrogel polymer content. The hydrogel system comprised a poly(N-isopropylacrylamide)-based thermogelling macromer (TGM) and a polyamidoamine crosslinker. The thermogelling macromer was copolymerized with (TGM/DBA) or without (TGM) a dimethyl-γ-butyrolactone acrylate (DBA)-containing lactone ring that modulated the lower critical solution temperature and thus, the hydrogel hydrophobicity, over time. Three hydrogel groups were examined: (1) 15wt.% TGM, (2) 15wt.% TGM/DBA, and (3) 20wt.% TGM/DBA. The hydrogels were implanted within an 8mm critical size rat cranial defect for 4 and 12weeks. Implants were harvested at each timepoint and analyzed for bone formation, hydrogel mineralization and tissue response using microcomputed tomography (microCT). Histology and fibrous capsule scoring showed a light inflammatory response at 4weeks that was mitigated by 12weeks for all groups. MicroCT scoring and bone volume quantification demonstrated a similar bone formation at 4weeks that was significantly increased for the more hydrophobic hydrogel formulations - 15wt.% TGM and 20wt.% TGM/DBA - from 4weeks to 12weeks. A complementary in vitro acellular mineralization study revealed that the hydrogels exhibited calcium binding properties in the presence of serum-containing media, which was modulated by the hydrogel hydrophobicity. The tailored mineralization capacity of these injectable, dual-gelling hydrogels with hydrolysis-dependent hydrophobicity presents an exciting property for their use in bone tissue engineering applications.

Use of porous space maintainers in staged mandibular reconstruction.

The success of mandibular reconstructions depends not only on restoring the form and function of lost bone but also on the preservation of the overlying soft tissue layer. In this case study, 5 porous polymethylmethacrylate space maintainers fabricated via patient-specific molds were implanted initially to maintain the vitality of the overlying oral mucosa during staged mandibular reconstructions. Three of the 5 patients healed well; the other 2 patients developed dehiscences, likely due to a thin layer of soft tissue overlying the implant. The results presented provide evidence that a larger investigation of space maintainers fabricated using this method is warranted.

Effects of antibiotic physicochemical properties on their release kinetics from biodegradable polymer microparticles.

PURPOSE: This study investigated the effects of the physicochemical properties of antibiotics on the morphology, loading efficiency, size, release kinetics, and antibiotic efficacy of loaded poly(DL-lactic-co-glycolic acid) (PLGA) microparticles (MPs) at different loading percentages. METHODS: Cefazolin, ciprofloxacin, clindamycin, colistin, doxycycline, and vancomycin were loaded at 10 and 20 wt% into PLGA MPs using a water-in-oil-in water double emulsion fabrication protocol. Microparticle morphology, size, loading efficiency, release kinetics, and antibiotic efficacy were assessed. RESULTS: The results from this study demonstrate that the chemical nature of loaded antibiotics, especially charge and molecular weight, influence the incorporation into and release of antibiotics from PLGA MPs. Drugs with molecular weights less than 600 Da displayed biphasic release while those with molecular weights greater than 1,000 Da displayed triphasic release kinetics. Large molecular weight drugs also had a longer delay before release than smaller molecular weight drugs. The negatively charged antibiotic cefazolin had lower loading efficiency than positively charged antibiotics. Microparticle size appeared to be mainly controlled by fabrication parameters, and partition and solubility coefficients did not appear to have an obvious effect on loading efficiency or release. Released antibiotics maintained their efficacy against susceptible strains over the duration of release. Duration of release varied between 17 and 49 days based on the type of antibiotic loaded. CONCLUSIONS: The data from this study indicate that the chemical nature of antibiotics affects properties of antibiotic-loaded PLGA MPs and allows for general prediction of loading and release kinetics.

Tissue response to composite hydrogels for vertical bone augmentation in the rat.

The objective of the present study was to develop a preclinical animal model for evaluating bone augmentation and to examine the level of bone augmentation induced by hydrogel composites. Design criteria outlined for the development of the animal model included rigid immobilization of bilateral implants apposed to the parietal bone of the rat, while avoiding the calvarial sutures. The animal model was evaluated through the implantation of hydrogel composites of oligo(poly(ethylene glycol) fumarate) (OPF) and gelatin microparticles releasing bone morphogenetic protein-2 (BMP-2). The BMP-2 release profile was varied and compared to the implantation of a material control without BMP-2. Each hydrogel composite was implanted within a polypropylene cassette, which was immobilized to the calvarial bone using screws, and empty cassettes were implanted as a control. The design criteria for the animal model were realized; however, the level of bone augmentation did not vary between any of the groups after 4 weeks. Osteoclastic bone resorption occurred to a higher extent in groups releasing BMP-2, but the cause could not be elucidated. In conclusion, a promising bone augmentation model was established in the rat; however, refinement of the hydrogel composites was suggested to optimize the constructs for bone augmentation applications.

Evaluation of antibiotic releasing porous polymethylmethacrylate space maintainers in an infected composite tissue defect model.

This study evaluated the in vitro and in vivo performance of antibiotic-releasing porous polymethylmethacrylate (PMMA)-based space maintainers comprising a gelatin hydrogel porogen and a poly(dl-lactic-co-glycolic acid) (PLGA) particulate carrier for antibiotic delivery. Colistin was released in vitro from either gelatin or PLGA microparticle loaded PMMA constructs, with gelatin-loaded constructs releasing colistin over approximately 7 days and PLGA microparticle-loaded constructs releasing colistin for up to 8 weeks. Three formulations with either burst release or extended release at different doses were tested in a rabbit mandibular defect inoculated with Acinetobacter baumannii (2×10(7) colony forming units ml(-1)). In addition, one material control that released antibiotic but was not inoculated with A. baumannii was tested. A. baumannii was not detectable in any animal after 12 weeks on culture of the defect, saliva, or blood. Defects with high dose extended release implants had greater soft tissue healing compared with defects with burst release implants, with 8 of 10 animals showing healed mucosae compared with 2 of 10 respectively. Extended release of locally delivered colistin via a PLGA microparticle carrier improved soft tissue healing compared with implants with burst release of colistin from a gelatin carrier.

Osteochondral tissue regeneration using a bilayered composite hydrogel with modulating dual growth factor release kinetics in a rabbit model.

Biodegradable oligo(poly(ethylene glycol) fumarate) (OPF) composite hydrogels have been investigated for the delivery of growth factors (GFs) with the aid of gelatin microparticles (GMPs) and stem cell populations for osteochondral tissue regeneration. In this study, a bilayered OPF composite hydrogel that mimics the distinctive hierarchical structure of native osteochondral tissue was utilized to investigate the effect of transforming growth factor-ß3 (TGF-ß3) with varying release kinetics and/or insulin-like growth factor-1 (IGF-1) on osteochondral tissue regeneration in a rabbit full-thickness osteochondral defect model. The four groups investigated included (i) a blank control (no GFs), (ii) GMP-loaded IGF-1 alone, (iii) GMP-loaded IGF-1 and gel-loaded TGF-ß3, and (iv) GMP-loaded IGF-1 and GMP-loaded TGF-ß3 in OPF composite hydrogels. The results of an in vitro release study demonstrated that TGF-ß3 release kinetics could be modulated by the GF incorporation method. At 12weeks post-implantation, the quality of tissue repair in both chondral and subchondral layers was analyzed based on quantitative histological scoring. All groups incorporating GFs resulted in a significant improvement in cartilage morphology compared to the control. Single delivery of IGF-1 showed higher scores in subchondral bone morphology as well as chondrocyte and glycosaminoglycan amount in adjacent cartilage tissue when compared to a dual delivery of IGF-1 and TGF-ß3, independent of the TGF-ß3 release kinetics. The results suggest that although the dual delivery of TGF-ß3 and IGF-1 may not synergistically enhance the quality of engineered tissue, the delivery of IGF-1 alone from bilayered composite hydrogels positively affects osteochondral tissue repair and holds promise for osteochondral tissue engineering applications.

Characterization of porous polymethylmethacrylate space maintainers for craniofacial reconstruction.

Porous polymethylmethacrylate (PMMA) has been used as an alloplastic bone substitute in the craniofacial complex, showing integration with the surrounding soft and hard tissue. This study investigated the physicochemical properties of curing and cured mixtures of a PMMA-based bone cement and a carboxymethylcellulose (CMC) gel porogen. Four formulations yielding porous PMMA of varied porosity were examined; specifically, two groups containing 30% (w/w) CMC gel in the mixture using a 7% (w/v) or 9% (w/v) stock CMC gel (30-7 and 30-9, respectively) and two groups containing 40% (w/w) CMC gel (40-7 and 40-9). An additional group comprising solid PMMA without CMC was investigated. The incorporation of the CMC gel into the PMMA bone cement during polymerization decreased the setting time from 608 ± 12 s for the solid PMMA to 427 ± 10 s for the 40-9 group, and decreased the maximum temperature from 81 ± 4°C for the solid PMMA to 38 ± 2°C for the 40-9 group. The porous PMMA groups exhibited reduced compressive strength and bending modulus and strength relative to the solid PMMA. All the porous PMMA formulations released more unconverted methylmethacrylate (MMA) monomer and N,N-dimethyl-p-toluidine (DMT) from cured specimens and less MMA and DMT from curing specimens than the solid PMMA. The data suggest that the physicochemical properties of the porous PMMA formulations are appropriate for their application in craniofacial space maintenance.

Evaluation of bone regeneration using the rat critical size calvarial defect.

Animal models that are reliably reproducible, appropriate analogs to the clinical condition they are used to investigate, and that offer minimal morbidity and periprocedural mortality to the subject, are the keystone to the preclinical development of translational technologies. For bone tissue engineering, a number of small animal models exist. Here we describe the protocol for one such model, the rat calvarial defect. This versatile model allows for evaluation of biomaterials and bone tissue engineering approaches within a reproducible, non-load-bearing orthotopic site. Crucial steps for ensuring appropriate experimental control and troubleshooting tips learned through extensive experience with this model are provided. The surgical procedure itself takes ∼30 min to complete, with ∼2 h of perioperative care, and tissue collection is generally performed 4-12 weeks postoperatively. Several analytical techniques are presented, which evaluate the cellular and extracellular matrix components, functionality and mineralization, including histological, mechanical and radiographic methods.

Imaging of poly(α-hydroxy-ester) scaffolds with X-ray phase-contrast microcomputed tomography.

Porous scaffolds based on poly(α-hydroxy-esters) are under investigation in many tissue engineering applications. A biological response to these materials is driven, in part, by their three-dimensional (3D) structure. The ability to evaluate quantitatively the material structure in tissue-engineering applications is important for the continued development of these polymer-based approaches. X-ray imaging techniques based on phase contrast (PC) have shown a tremendous promise for a number of biomedical applications owing to their ability to provide a contrast based on alternative X-ray properties (refraction and scatter) in addition to X-ray absorption. In this research, poly(α-hydroxy-ester) scaffolds were synthesized and imaged by X-ray PC microcomputed tomography. The 3D images depicting the X-ray attenuation and phase-shifting properties were reconstructed from the measurement data. The scaffold structure could be imaged by X-ray PC in both cell culture conditions and within the tissue. The 3D images allowed for quantification of scaffold properties and automatic segmentation of scaffolds from the surrounding hard and soft tissues. These results provide evidence of the significant potential of techniques based on X-ray PC for imaging polymer scaffolds.

In situ formation of porous space maintainers in a composite tissue defect.

Reconstruction of composite defects involving bone and soft tissue presents a significant clinical challenge. In the craniofacial complex, reconstruction of the soft and hard tissues is critical for both functional and aesthetic outcomes. Constructs for space maintenance provide a template for soft tissue regeneration, priming the wound bed for a definitive repair of the bone tissue with greater success. However, materials used clinically for space maintenance are subject to poor soft tissue integration, which can result in wound dehiscence. Porous materials in space maintenance applications have been previously shown to support soft tissue integration and to allow for drug release from the implant to further prepare the wound bed for definitive repair. This study evaluated solid and low porosity (16.9% ± 4.1%) polymethylmethacrylate space maintainers fabricated intraoperatively and implanted in a composite rabbit mandibular defect model for 12 weeks. The data analyses showed no difference in the solid and porous groups both histologically, evaluating the inflammatory response at the interface and within the pores of the implants, and grossly, observing the healing of the soft tissue defect over the implant. These results demonstrate the potential of porous polymethylmethacrylate implants formed in situ for space maintenance in the craniofacial complex, which may have implications in the potential delivery of therapeutic drugs to prime the wound site for a definitive bone repair.

Harnessing and modulating inflammation in strategies for bone regeneration.

Inflammation is an immediate response that plays a critical role in healing after fracture or injury to bone. However, in certain clinical contexts, such as in inflammatory diseases or in response to the implantation of a biomedical device, the inflammatory response may become chronic and result in destructive catabolic effects on the bone tissue. Since our previous review 3 years ago, which identified inflammatory signals critical for bone regeneration and described the inhibitory effects of anti-inflammatory agents on bone healing, a multitude of studies have been published exploring various aspects of this emerging field. In this review, we distinguish between regenerative and damaging inflammatory processes in bone, update our discussion of the effects of anti-inflammatory agents on bone healing, summarize recent in vitro and in vivo studies demonstrating how inflammation can be modulated to stimulate bone regeneration, and identify key future directions in the field.

Antibiotic-releasing porous polymethylmethacrylate/gelatin/antibiotic constructs for craniofacial tissue engineering.

An antibiotic-releasing porous polymethylmethacrylate (PMMA) construct was developed to maintain the bony space and prime the wound site in the initial step of a two-stage regenerative medicine approach toward reconstructing significant bony or composite craniofacial tissue defects. Porous PMMA constructs incorporating gelatin microparticles (GMPs) were fabricated by the sequential assembly of GMPs, the antibiotic colistin, and a clinically used bone cement formulation of PMMA powder and methylmethacrylate liquid. PMMA/gelatin/antibiotic constructs with varying gelatin incorporation and drug content were investigated to elucidate the relationship between material composition and construct properties (porosity and drug release kinetics). The porosity of PMMA/gelatin/antibiotic constructs ranged between 7.6±1.8% and 38.4±1.4% depending on the amount of gelatin incorporated and the drug solution added for gelatin swelling. The constructs released colistin over 10 or 14 days with an average release rate per day above 10 µg/ml. The porosity and in vitro colistin release kinetics of PMMA/gelatin/antibiotic constructs were tuned by varying the material composition and fabrication parameters. This study demonstrates the potential of gelatin-incorporating PMMA constructs as a functional space maintainer for both promoting tissue healing/coverage and addressing local infections, enabling better long-term success of the definitive regenerated tissue construct.

Delivery of plasmid DNA encoding bone morphogenetic protein-2 with a biodegradable branched polycationic polymer in a critical-size rat cranial defect model.

This study investigated the delivery of plasmid DNA (pDNA) encoding bone morphogenetic protein-2 in the form of polyplexes with a biodegradable branched triacrylate/amine polycationic polymer (TAPP) that were complexed with gelatin microparticles (GMPs) loaded within a porous tissue engineering scaffold. More specifically, the study investigated the interplay between TAPP degradation, gelatin degradation, pDNA release, and bone formation in a critical-size rat cranial defect model. The pDNA release kinetics in vitro were not affected by the crosslinking density of the GMPs but depended, rather, on the degradation rates of the TAPPs. Besides the initial release of polyplexes not bound to the GMPs and the minimal release of polyplexes through diffusion or dissociation from the GMPs, the pDNA was likely released as naked pDNA or as part of an incomplete polyplex, after the degradation of fragments of the polycationic polymer. After 30 days, significantly higher amounts of pDNA were released (93%-98%) from composite scaffolds containing naked pDNA or pDNA complexed with P-AEPZ (synthesized with 1-[2-aminoethyl]piperazine, a faster degrading TAPP) compared with those containing pDNA complexed with P-DED (synthesized with N,N-dimethylethylenediamine, a slower degrading TAPP) (74%-82%). Composite scaffolds containing GMPs complexed with TAPP/pDNA polyplexes did not result in enhanced bone formation, as analyzed by microcomputed tomography and histology, in a critical-size rat cranial defect at 12 weeks postimplantation compared with those loaded with naked pDNA. The results demonstrate that polycationic polymers with a slow degradation rate can prolong the release of pDNA from the composite scaffolds and suggest that a gene delivery system comprising biodegradable polycationic polymers should be designed to release the pDNA in an intact polyplex form.

Uncultured marrow mononuclear cells delivered within fibrin glue hydrogels to porous scaffolds enhance bone regeneration within critical-sized rat cranial defects.

For bone tissue engineering, the benefits of incorporating mesenchymal stem cells (MSCs) into porous scaffolds are well established. There is, however, little consensus on the effects of or need for MSC handling ex vivo. Culture and expansion of MSCs adds length and cost, and likely increases risk associated with treatment. We evaluated the effect of using uncultured bone marrow mononuclear cells (bmMNCs) encapsulated within fibrin glue hydrogels and seeded into porous scaffolds to regenerate bone over 12 weeks in an 8-mm-diameter, critical-sized rat cranial defect. A full factorial experimental design was used to evaluate bone formation within model poly(L-lactic acid) and corraline hydroxyapatite scaffolds with or without platelet-rich plasma (PRP) and bmMNCs. Mechanical push-out testing, microcomputed tomographical analyses, and histology were performed. PRP showed no benefit for bone formation. Cell-laden poly(L-lactic acid) scaffolds without PRP required significantly greater force to displace from surrounding tissues than control (cell-free) scaffolds, but no differences were observed during push-out testing of coral scaffolds. For bone volume formation as analyzed by microcomputed tomography, significant positive overall effects were observed with bmMNC incorporation. These data suggest that bmMNCs may provide therapeutic advantages in bone tissue engineering applications without the need for culture, expansion, and purification.

Fibrin glue as a drug delivery system.

Fibrin glue has been used surgically for decades for hemostasis as well as a sealant. It has also been researched as both a gel for cell delivery and a vehicle for drug delivery. The drug delivery applications for fibrin glue span tissue engineering to chemotherapy and involve several mechanisms for drug matrix interactions and control of release kinetics. Additionally, drugs or factors can be loaded in the gel via impregnation and tethering to the gel through covalent linkages or affinity-based systems. This review highlights recent research of fibrin glue as a drug delivery vehicle.

Evaluation of soft tissue coverage over porous polymethylmethacrylate space maintainers within nonhealing alveolar bone defects.

Current treatment of traumatic craniofacial injuries often involves early free tissue transfer, even if the recipient site is contaminated or lacks soft tissue coverage. There are no current tissue engineering strategies to definitively regenerate tissues in such an environment at an early time point. For a tissue engineering approach to be employed in the treatment of such injuries, a two-stage approach could potentially be used. The present study describes methods for fabrication, characterization, and processing of porous polymethylmethacrylate (PMMA) space maintainers for temporary retention of space in bony craniofacial defects. Carboxymethylcellulose hydrogels were used as a porogen. Implants with controlled porosity and pore interconnectivity were fabricated by varying the ratio of hydrogel:polymer and the amount of carboxymethylcellulose within the hydrogel. The in vivo tissue response to the implants was observed by implanting solid, low-porosity, and high-porosity implants (n = 6) within a nonhealing rabbit mandibular defect that included an oral mucosal defect to allow open communication between the oral cavity and the mandibular defect. Oral mucosal wound healing was observed after 12 weeks and was complete in 3/6 defects filled with solid PMMA implants and 5/6 defects filled with either a low- or high-porosity PMMA implant. The tissue response around and within the pores of the two formulations of porous implants tested in vivo was characterized, with the low-porosity implants surrounded by a minimal but well-formed fibrous capsule in contrast to the high-porosity implants, which were surrounded and invaded by almost exclusively inflammatory tissue. On the basis of these results, PMMA implants with limited porosity hold promise for temporary implantation and space maintenance within clean/contaminated bone defects.

In vitro cytotoxicity of single-walled carbon nanotube/biodegradable polymer nanocomposites.

Injectable nanocomposites made of biodegradable poly(propylene fumarate) and the crosslinking agent propylene fumarate-diacrylate as well as each of three forms of single-walled carbon nanotubes (SWNTs) were evaluated for their in vitro cytotoxicity. Unreacted components, crosslinked networks, and degradation products of the nanocomposites were investigated for their effects on cell viability using a fibroblast cell line in vitro. The results did not reveal any in vitro cytotoxicity for purified SWNTs, SWNTs functionalized with 4-tert-butylphenylene, and ultra-short SWNTs at 1- 100 microg/mL concentrations. Moreover, nearly 100% cell viability was observed on all crosslinked nanocomposites and cell attachment on their surfaces was comparable with that on tissue culture polystyrene. The degradation products of the nanocomposites displayed a dose-dependent adverse effect on cells, which was partially due to increased osmolarity by the conditions of accelerated degradation and could be overcome at diluted concentrations. These results demonstrate that all three tested nanocomposites have favorable cytocompatibility for potential use as scaffolds for bone tissue engineering applications.

Injectable in situ cross-linkable nanocomposites of biodegradable polymers and carbon nanostructures for bone tissue engineering.

This study investigates the effects of nanostructure size and surface area on the rheological properties of un-cross-linked poly(propylene fumarate) (PPF) nanocomposites and the mechanical properties of cross-linked nanocomposites as a function of the nanostructure loading. Three model carbon nanostructures were examined, C(60) fullerenes, ultra-short single-walled carbon nanotubes (US-tubes) and single-walled carbon nanotubes (SWNTs). Rheological measurements showed that C60 and US-tube un-cross-linked nanocomposites exhibited viscous-like characteristics with the complex viscosity independent of frequency for nanostructure concentrations up to 1 wt%. Compressive and flexural mechanical testing demonstrated significant mechanical reinforcement of US-tube and SWNT nanocomposites as compared to cross-linked polymer alone, with an up to twofold increase in the mechanical properties. Scanning electron microscopy examination of the fracture surface of cross-linked US-tube nanocomposite revealed lack of aggregation of US-tubes. Although sol fraction studies did not provide any evidence of additional cross-linking, due to the presence of US-tubes in the nanocomposites, transmission electron microscopy studies suggested the crystallization of PPF on the surface of US-tubes which can contribute to the mechanical reinforcement of the US-tube nanocomposites. These results demonstrate that the rheological properties of un-cross-linked nanocomposites depend mainly on the carbon nanostructure size, whereas the mechanical properties of the cross-linked nanocomposites are dependent on the carbon nanostructure surface area. The data also suggest that US-tube nanocomposites are suitable for further consideration as injectable scaffolds for bone tissue engineering applications.