Nanofiber orientation and surface functionalization modulate human mesenchymal stem cell behavior in vitro.

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ε-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration.

Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration.

Effects of in vivo mechanical loading on large bone defect regeneration.

Fracture healing is highly sensitive to mechanical conditions; however, the effects of mechanical loading on large bone defect regeneration have not been evaluated. In this study, we investigated the effects of functional loading on repair of critically sized segmental bone defects. About 6-mm defects were created in rat femora, and each defect received 5 µg recombinant human bone morphogenetic protein-2 (rhBMP-2), delivered in alginate hydrogel. Limbs were stabilized by either stiff fixation plates for the duration of the study or compliant plates that allowed transfer of compressive ambulatory loads beginning at week 4. Healing was assessed by digital radiography, microcomputed tomography, mechanical testing, histology, and finite element modeling. Loading significantly increased regenerate bone volume and average polar moment of inertia. The response to loading was location-dependent with the polar moment of inertia increased at the proximal end of the defect but not the distal end. As a result, torsional stiffness was 58% higher in the compliant plate group, but failure torque was not altered. In single samples assessed for histology from each group, a qualitatively greater amount of cartilage and a lesser degree of remodeling to lamellar bone occurred in the loaded group compared to the stiff plate group. Finally, principal strain histograms, calculated by FE modeling, revealed that the compliant plate samples had adapted to more efficiently distribute loads in the defects. Together, these data demonstrate that functional transfer of axial loads alters BMP-induced large bone defect repair by increasing the amount and distribution of bone formed within the defect.

Spatiotemporal delivery of bone morphogenetic protein enhances functional repair of segmental bone defects.

Osteogenic growth factors that promote endogenous repair mechanisms hold considerable potential for repairing challenging bone defects. The local delivery of one such growth factor, bone morphogenetic protein (BMP), has been successfully translated to clinical practice for spinal fusion and bone fractures. However, improvements are needed in the spatial and temporal control of BMP delivery to avoid the currently used supraphysiologic doses and the concomitant adverse effects. We have recently introduced a hybrid protein delivery system comprised of two parts: a perforated nanofibrous mesh that spatially confines the defect region and a functionalized alginate hydrogel that provides temporal growth factor release kinetics. Using this unique spatiotemporal delivery system, we previously demonstrated BMP-mediated functional restoration of challenging 8mm femoral defects in a rat model. In this study, we compared the efficacy of the hybrid system in repairing segmental bone defects to that of the current clinical standard, collagen sponge, at the same dose of recombinant human BMP-2. In addition, we investigated the specific role of the nanofibrous mesh tube on bone regeneration. Our results indicate that the hybrid delivery system significantly increased bone regeneration and improved biomechanical function compared to collagen sponge delivery. Furthermore, we observed that presence of the nanofiber mesh tube was essential to promote maximal mineralized matrix synthesis, prevent extra-anatomical mineralization, and guide an integrated pattern of bone formation. Together, these results suggest that spatiotemporal strategies for osteogenic protein delivery may enhance clinical outcomes by improving localized protein retention.

Effects of protein dose and delivery system on BMP-mediated bone regeneration.

Delivery of recombinant proteins is a proven therapeutic strategy to promote endogenous repair mechanisms and tissue regeneration. Bone morphogenetic protein-2 (rhBMP-2) has been used to promote spinal fusion and repair of challenging bone defects; however, the current clinically-used carrier, absorbable collagen sponge, requires high doses and has been associated with adverse complications. We evaluated the hypothesis that the relationship between protein dose and regenerative efficacy depends on delivery system. First, we determined the dose-response relationship for rhBMP-2 delivered to 8-mm rat bone defects in a hybrid nanofiber mesh/alginate delivery system at six doses ranging from 0 to 5 µg. Next, we directly compared the hybrid delivery system to the collagen sponge at 0.1 and 1.0 µg. Finally, we compared the in vivo protein release properties of the two delivery methods. In the hybrid delivery system, bone volume, connectivity and mechanical properties increased in a dose-dependent manner to rhBMP-2. Consistent bridging of the defect was observed for doses of 1.0 µg and greater. Compared to collagen sponge delivery at the same 1.0 µg dose, the hybrid system yielded greater connectivity by week 4 and 2.5-fold greater bone volume by week 12. These differences may be explained by the significantly greater protein retention in the hybrid system compared to collagen sponge. This study demonstrates a clear dose-dependent effect of rhBMP-2 delivered using a hybrid nanofiber mesh/alginate delivery system. Furthermore, the effective dose was found to vary with delivery system, demonstrating the importance of biomaterial carrier properties in the delivery of recombinant proteins.

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects.

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal µ-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. µ-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

Colonization and osteogenic differentiation of different stem cell sources on electrospun nanofiber meshes.

Numerous challenges remain in the successful clinical translation of cell-based therapies for musculoskeletal tissue repair, including the identification of an appropriate cell source and a viable cell delivery system. The aim of this study was to investigate the attachment, colonization, and osteogenic differentiation of two stem cell types, human mesenchymal stem cells (hMSCs) and human amniotic fluid stem (hAFS) cells, on electrospun nanofiber meshes. We demonstrate that nanofiber meshes are able to support these cell functions robustly, with both cell types demonstrating strong osteogenic potential. Differences in the kinetics of osteogenic differentiation were observed between hMSCs and hAFS cells, with the hAFS cells displaying a delayed alkaline phosphatase peak, but elevated mineral deposition, compared to hMSCs. We also compared the cell behavior on nanofiber meshes to that on tissue culture plastic, and observed that there is delayed initial attachment and proliferation on meshes, but enhanced mineralization at a later time point. Finally, cell-seeded nanofiber meshes were found to be effective in colonizing three-dimensional scaffolds in an in vitro system. This study provides support for the use of the nanofiber mesh as a model surface for cell culture in vitro, and a cell delivery vehicle for the repair of bone defects in vivo.

In vivo model for evaluating the effects of mechanical stimulation on tissue-engineered bone repair.

It has long been known that the bone adapts according to the local mechanical environment. To date, however, a model for studying the effects of functional mechanical loading on tissue-engineered bone repair in vivo has not yet been established. We have developed a rat femoral defect model, in which ambulatory loads are transduced through the implanted tissue-engineered construct to elucidate the role of the mechanical environment in functional restoration of a large bone defect. This model uses compliant fixation plates with integrated elastomeric segments, which allow transduction of ambulatory loads. Multiaxially and uniaxially compliant plates were characterized by mechanical testing and evaluated using in vivo pilot studies. In the first study, experimental limbs were implanted with multiaxial plates, which have a low stiffness in multiple loading modes. In the second study, experimental limbs were stabilized by a uniaxial plate, which allowed only axial deformation of the defect. X-ray scans and mechanical testing revealed that the multiaxial plates were insufficient to stabilize the defect and prevent fracture under ambulatory loads as a result of low flexural and torsional stiffness. The uniaxial plates, however, maintained integrity of the defect when implanted over a 12 week period. Postmortem microCT scans revealed a 19% increase in bone volume in the axially loaded limb compared with the contralateral standard control, and postmortem mechanical testing indicated that torsional strength and stiffness were increased 25.6- and 3.9-fold, respectively, compared with the control. Finite element modeling revealed high strain gradients in the soft tissue adjacent to the newly formed bone within the implanted construct. This study introduces an in vivo model for studying the effects of physiological mechanical loading on tissue-engineered bone repair. Preliminary results using this new in vivo model with the uniaxially compliant plate showed positive effects of load-bearing on functional defect repair.

Theoretical analysis of engineered cartilage oxygenation: influence of construct thickness and media flow rate.

A novel parallel-plate bioreactor has been shown to modulate the mechanical and biochemical properties of engineered cartilage by the application of fluid-induced shear stress. Flow or perfusion bioreactors may improve tissue development via enhanced transport of nutrients or gases as well as the application of mechanical stimuli, or a combination of these factors. The goal of this study was to complement observed experimental responses to flow by simulating oxygen transport within cartilage constructs of different thicknesses (250 microm or 1 mm). Using numerical computation of convection-diffusion equations, the evaluation of the tissue oxygenation is performed. Four culture conditions are defined based on tissue thickness and flow rates ranging from 0 to approximately 25 mL min(-1). Under these experimental conditions results show a mean oxygen concentration within the tissue varying from 0.01 to 0.19 mol m(-3) as a function of the tissue thickness and the magnitude of the applied shear stress. More generally, the influence of shear stress varying (via flow rate modification) from 10(-3) to 10 dynes cm(-2) on the tissue oxygenation is studied. The influence on the results of important physical parameters such as the maximal oxygen consumption rate of cells is discussed. Lastly, the importance of oxygen concentration in the lower chamber and its relevance to tissue oxygenation are highlighted by the model results.

Chondrogenic differentiation of amniotic fluid-derived stem cells.

For regenerating damaged articular cartilage, it is necessary to identify an appropriate cell source that is easily accessible, can be expanded to large numbers, and has chondrogenic potential. Amniotic fluid-derived stem (AFS) cells have recently been isolated from human and rodent amniotic fluid and shown to be highly proliferative and broadly pluripotent. The purpose of this study was to investigate the chondrogenic potential of human AFS cells in pellet and alginate hydrogel cultures. Human AFS cells were expanded in various media conditions, and cultured for three weeks with growth factor supplementation. There was increased production of sulfated glycosaminoglycan (sGAG) and type II collagen in response to transforming growth factor-beta (TGF-beta) supplementation, with TGF-beta1 producing greater increases than TGF-beta3. Modification of expansion media supplements and addition of insulin-like growth factor-1 during pellet culture further increased sGAG/DNA over TGF-beta1 supplementation alone. Compared to bone marrow-derived mesenchymal stem cells, the AFS cells produced less cartilaginous matrix after three weeks of TGF-beta1 supplementation in pellet culture. Even so, this study demonstrates that AFS cells have the potential to differentiate along the chondrogenic lineage, thus establishing the feasibility of using these cells for cartilage repair applications.