Effects of osteogenic ambulatory mechanical stimulation on early stages of BMP-2 mediated bone repair.

Purpose: Mechanical loading of bone defects through rehabilitation is a promising approach to stimulate repair and reduce nonunion risk; however, little is known about how therapeutic mechanical stimuli modulate early-stage repair before mineralized bone formation. The objective of this study was to investigate the early effects of osteogenic loading on cytokine expression and angiogenesis during the first 3 weeks of BMP-2 mediated segmental bone defect repair.Materials and Methods: A rat model of BMP-2 mediated bone defect repair was subjected to an osteogenic mechanical loading protocol using ambulatory rehabilitation and a compliant, load-sharing fixator with an integrated implantable strain sensor. The effect of fixator load-sharing on local tissue strain, angiogenesis, and cytokine expression was evaluated.Results: Using sensor readings for local measurements of boundary conditions, finite element simulations showed strain became amplified in remaining soft tissue regions between 1 and 3 weeks (Week 3: load-sharing: -1.89 ± 0.35% and load-shielded: -1.38 ± 0.35% vs. Week 1: load-sharing: -1.54 ± 0.17%; load-shielded: -0.76 ± 0.06%). Multivariate analysis of cytokine arrays revealed that load-sharing significantly altered expression profiles in the defect tissue at 2 weeks compared to load-shielded defects. Specifically, loading reduced VEGF (p = 0.052) and increased CXCL5 (LIX) levels. Subsequently, vascular volume in loaded defects was reduced relative to load-shielded defects but similar to intact bone at 3 weeks. Endochondral bone repair was also observed histologically in loaded defects at 3 weeks.Conclusions: Together, these results demonstrate that moderate ambulatory strains previously shown to stimulate bone regeneration significantly alter early angiogenic and cytokine signaling and may promote endochondral ossification.

Implantable Sensors for Regenerative Medicine.

The translation of many tissue engineering/regenerative medicine (TE/RM) therapies that demonstrate promise in vitro are delayed or abandoned due to reduced and inconsistent efficacy when implemented in more complex and clinically relevant preclinical in vivo models. Determining mechanistic reasons for impaired treatment efficacy is challenging after a regenerative therapy is implanted due to technical limitations in longitudinally measuring the progression of key environmental cues in vivo. The ability to acquire real-time measurements of environmental parameters of interest including strain, pressure, pH, temperature, oxygen tension, and specific biomarkers within the regenerative niche in situ would significantly enhance the information available to tissue engineers to monitor and evaluate mechanisms of functional healing or lack thereof. Continued advancements in material and fabrication technologies utilized by microelectromechanical systems (MEMSs) and the unique physical characteristics of passive magnetoelastic sensor platforms have created an opportunity to implant small, flexible, low-power sensors into preclinical in vivo models, and quantitatively measure environmental cues throughout healing. In this perspective article, we discuss the need for longitudinal measurements in TE/RM research, technical progress in MEMS and magnetoelastic approaches to implantable sensors, the potential application of implantable sensors to benefit preclinical TE/RM research, and the future directions of collaborative efforts at the intersection of these two important fields.

Wireless Implantable Sensor for Noninvasive, Longitudinal Quantification of Axial Strain Across Rodent Long Bone Defects.

Bone development, maintenance, and regeneration are remarkably sensitive to mechanical cues. Consequently, mechanical stimulation has long been sought as a putative target to promote endogenous healing after fracture. Given the transient nature of bone repair, tissue-level mechanical cues evolve rapidly over time after injury and are challenging to measure noninvasively. The objective of this work was to develop and characterize an implantable strain sensor for noninvasive monitoring of axial strain across a rodent femoral defect during functional activity. Herein, we present the design, characterization, and in vivo demonstration of the device's capabilities for quantitatively interrogating physiological dynamic strains during bone regeneration. Ex vivo experimental characterization of the device showed that it possessed promising sensitivity, signal resolution, and electromechanical stability for in vivo applications. The digital telemetry minimized power consumption, enabling extended intermittent data collection. Devices were implanted in a rat 6 mm femoral segmental defect model, and after three days, data were acquired wirelessly during ambulation and synchronized to corresponding radiographic videos, validating the ability of the sensor to noninvasively measure strain in real-time. Together, these data indicate the sensor is a promising technology to quantify tissue mechanics in a specimen specific manner, facilitating more detailed investigations into the role of the mechanical environment in dynamic bone healing and remodeling processes.

Characterization of Cell-Type-Specific Drug Transport and Resistance of Breast Cancers Using Tumor-Microenvironment-on-Chip.

Heterogeneous response and resistance of cancer cells to chemotherapeutic drugs pose a significant challenge for successful cancer treatments. In this study, an integrated experimental and theoretical analysis of cellular drug transport was developed. The experimental platform, called tumor-microenvironment-on-chip (T-MOC), is a microfluidic platform where cancer cells were cultured within a three-dimensional extracellular matrix perfused with interstitial fluid. Three types of human breast cancer cell lines (MCF-7, MDA-MB-231, and SUM-159PT) were cultured on this T-MOC platform, and their drug response and resistance to doxorubicin were characterized by time-lapse quantitative fluorescence microscopy. To study the effects of nanoparticle-mediated drug delivery, the transport and action of doxorubicin encapsulated nanoparticles were also examined. Based on the experimental data obtained, a theoretical model was developed to quantify and ultimately predict the cellular transport processes of drugs cell-type specifically. The results demonstrate that the cellular drug transport can be cell-type-specifically quantified by rate constants representing the uptake and efflux of doxorubicin across the cellular membrane.

Triple growth factor delivery promotes functional bone regeneration following composite musculoskeletal trauma.

Successful bone healing in severe trauma depends on early revascularization to restore oxygen, nutrient, growth factor, and progenitor cell supply to the injury. Therapeutic angiogenesis strategies have therefore been investigated to promote revascularization following severe bone injuries; however, results have been inconsistent. This is the first study investigating the effects of dual angiogenic growth factors (VEGF and PDGF) with low-dose bone morphogenetic protein-2 (BMP-2; 2.5 µg) on bone healing in a clinically challenging composite bone-muscle injury model. Our hydrogel-based delivery systems demonstrated a more than 90% protein entrapment efficiency and a controlled simultaneous release of three growth factors over 28 days. Co-stimulation of microvascular fragment constructs with VEGF and PDGF promoted vascular network formation in vitro compared to VEGF or PDGF alone. In an in vivo model of segmental bone and volumetric muscle loss injury, combined VEGF (5 µg) and PDGF (7.5 µg or 15 µg) delivery with a low dose of BMP-2 significantly enhanced regeneration of vascularized bone compared to BMP-2 treatment alone. Notably, the regenerated bone mechanics reached ~60% of intact bone, a value that was previously only achieved by delivery of high-dose BMP-2 (10 µg) in this injury model. Overall, sustained delivery of VEGF, PDFG, and BMP-2 is a promising strategy to promote functional vascularized bone tissue regeneration following severe composite musculoskeletal injury. Although this study is conducted in a clinically relevant composite injury model in rats using a simultaneous release strategy, future studies are necessary to test the regenerative potential of spatiotemporally controlled delivery of triple growth factors on bone healing using large animal models. STATEMENT OF SIGNIFICANCE: Volumetric muscle loss combined with delayed union or non-union bone defect causes deleterious effects on bone regeneration even with the supplementation of bone morphogenetic protein-2 (BMP-2). In this study, the controlled delivery of dual angiogenic growth factors (vascular endothelial growth factor [VEGF] + Platelet-derived growth factor [PDGF]) increases vascular growth in vitro. Co-delivering VEGF+PDGF significantly increase the bone formation efficacy of low-dose BMP-2 and improves the mechanics of regenerated bone in a challenging composite bone-muscle injury model.

Wireless sensor enables longitudinal monitoring of regenerative niche mechanics during rehabilitation that enhance bone repair.

Mechanical loads exerted on the skeleton during activities such as walking are important regulators of bone repair, but dynamic biomechanical signals are difficult to measure inside the body. The inability to measure the mechanical environment in injured tissues is a significant barrier to developing integrative regenerative and rehabilitative strategies that can accelerate recovery from fracture, segmental bone loss, and spinal fusion. Here we engineered an implantable strain sensor platform and longitudinally measured strain across a bone defect in real-time throughout rehabilitation. The results showed that load-sharing permitted by a load-sharing fixator initially delivered a two-fold increase in deformation magnitude, subsequently increased mineralized bridging by nearly three-fold, and increased bone formation by over 60%. These data implicate a critical role for early mechanical cues on the long term healing response as strain cycle magnitude at 1 week (before appreciable healing occurred) had a significant positive correlation with the long-term bone regeneration outcomes. Furthermore, we found that sensor readings correlated with the status of healing, suggesting a role for strain sensing as an X-ray-free healing assessment platform. Therefore, non-invasive strain measurements may possess diagnostic potential to evaluate bone repair and reduce clinical reliance on current radiation-emitting imaging methods. Together, this study demonstrates a promising framework to quantitatively develop and exploit mechanical rehabilitation strategies that enhance bone repair.

Impaction durability of porous polyether-ether-ketone (PEEK) and titanium-coated PEEK interbody fusion devices.

BACKGROUND CONTEXT: Various surface modifications, often incorporating roughened or porous surfaces, have recently been introduced to enhance osseointegration of interbody fusion devices. However, these topographical features can be vulnerable to damage during clinical impaction. Despite the potential negative impact of surface damage on clinical outcomes, current testing standards do not replicate clinically relevant impaction loading conditions. PURPOSE: The purpose of this study was to compare the impaction durability of conventional smooth polyether-ether-ketone (PEEK) cervical interbody fusion devices with two surface-modified PEEK devices that feature either a porous structure or plasma-sprayed titanium coating. STUDY DESIGN/SETTING: A recently developed biomechanical test method was adapted to simulate clinically relevant impaction loading conditions during cervical interbody fusion procedures. METHODS: Three cervical interbody fusion devices were used in this study: smooth PEEK, plasma-sprayed titanium-coated PEEK, and porous PEEK (n=6). Following Kienle et al., devices were impacted between two polyurethane blocks mimicking vertebral bodies under a constant 200 N preload. The posterior tip of the device was placed at the entrance between the polyurethane blocks, and a guided 1-lb weight was impacted upon the anterior face with a maximum speed of 2.6 m/s to represent the strike force of a surgical mallet. Impacts were repeated until the device was fully impacted. Porous PEEK durability was assessed using micro-computed tomography (µCT) pre- and postimpaction. Titanium-coating coverage pre- and postimpaction was assessed using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy. Changes to the surface roughness of smooth and titanium-coated devices were also evaluated. RESULTS: Porous PEEK and smooth PEEK devices showed minimal macroscopic signs of surface damage, whereas the titanium-coated devices exhibited substantial visible coating loss. Quantification of the porous PEEK deformation demonstrated that the porous structure maintained a high porosity (>65%) following impaction that would be available for bone ingrowth, and exhibited minimal changes to pore size and depth. SEM and energy dispersive X-ray spectroscopy analysis of titanium-coated devices demonstrated substantial titanium coating loss after impaction that was corroborated with a decrease in surface roughness. Smooth PEEK showed minimal signs of damage using SEM, but demonstrated a decrease in surface roughness. CONCLUSION: Although recent surface modifications to interbody fusion devices are beneficial for osseointegration, they may be susceptible to damage and wear during impaction. The current study found porous PEEK devices to show minimal damage during simulated cervical impaction, whereas titanium-coated PEEK devices lost substantial titanium coverage.

Effect of porous orthopaedic implant material and structure on load sharing with simulated bone ingrowth: A finite element analysis comparing titanium and PEEK.

Osseointegration of load-bearing orthopaedic implants, including interbody fusion devices, is critical to long-term biomechanical functionality. Mechanical loads are a key regulator of bone tissue remodeling and maintenance, and stress-shielding due to metal orthopaedic implants being much stiffer than bone has been implicated in clinical observations of long-term bone loss in tissue adjacent to implants. Porous features that accommodate bone ingrowth have improved implant fixation in the short term, but long-term retrieval studies have sometimes demonstrated limited, superficial ingrowth into the pore layer of metal implants and aseptic loosening remains a problem for a subset of patients. Polyether-ether-ketone (PEEK) is a widely used orthopaedic material with an elastic modulus more similar to bone than metals, and a manufacturing process to form porous PEEK was recently developed to allow bone ingrowth while preserving strength for load-bearing applications. To investigate the biomechanical implications of porous PEEK compared to porous metals, we analyzed finite element (FE) models of the pore structure-bone interface using two clinically available implants with high (> 60%) porosity, one being constructed from PEEK and the other from electron beam 3D-printed titanium (Ti). The objective of this study was to investigate how porous PEEK and porous Ti mechanical properties affect load sharing with bone within the porous architectures over time. Porous PEEK substantially increased the load share transferred to ingrown bone compared to porous Ti under compression (i.e. at 4 weeks: PEEK = 66%; Ti = 13%), tension (PEEK = 71%; Ti = 12%), and shear (PEEK = 68%; Ti = 9%) at all time points of simulated bone ingrowth. Applying PEEK mechanical properties to the Ti implant geometry and vice versa demonstrated that the observed increases in load sharing with PEEK were primarily due to differences in intrinsic elastic modulus and not pore architecture (i.e. 4 weeks, compression: PEEK material/Ti geometry = 53%; Ti material/PEEK geometry = 12%). Additionally, local tissue energy effective strains on bone tissue adjacent to the implant under spinal load magnitudes were over two-fold higher with porous PEEK than porous Ti (i.e. 4 weeks, compression: PEEK = 784 ± 351 microstrain; Ti = 180 ± 300 microstrain; and 12 weeks, compression: PEEK = 298 ± 88 microstrain; Ti = 121 ± 49 microstrain). The higher local strains on bone tissue in the PEEK pore structure were below previously established thresholds for bone damage but in the range necessary for physiological bone maintenance and adaptation. Placing these strain magnitudes in the context of literature on bone adaptation to mechanical loads, this study suggests that porous PEEK structures may provide a more favorable mechanical environment for bone formation and maintenance under spinal load magnitudes than currently available porous 3D-printed Ti, regardless of the level of bone ingrowth.

Delivery vehicle effects on bone regeneration and heterotopic ossification induced by high dose BMP-2.

Bone morphogenetic protein-2 (BMP-2), delivered on absorbable collagen sponge, is frequently used to treat bone defects. However, supraphysiological BMP-2 doses are common and often associated with complications such as heterotopic ossification and inflammation, causing pain and impaired mobility. This has prompted investigations into strategies to spatially control bone regeneration, for example growth factor delivery in appropriate scaffolds. Our objective was to investigate the spatiotemporal effects of high dose BMP-2 on bone regeneration as a function of the delivery vehicle. We hypothesized that an alginate delivery system would spatially restrict bone formation compared to a collagen sponge delivery system. In vitro, BMP-2 release was accelerated from collagen sponge compared to alginate constructs. In vivo, bone regeneration was evaluated over 12weeks in critically sized rat femoral segmental defects treated with 30µg rhBMP-2 in alginate hydrogel or collagen sponge, surrounded by perforated nanofiber meshes. Total bone volume, calculated from micro-CT reconstructions, was higher in the alginate group at 12weeks. Though bone volume within the central defect region was greater in the alginate group at 8 and 12weeks, heterotopic bone volume was similar between groups. Likewise, mechanical properties from ex vivo torsional testing were comparable between groups. Histology corroborated these findings and revealed heterotopic mineralization at 2weeks post-surgery in both groups. Overall, this study recapitulated the heterotopic ossification associated with high dose BMP-2 delivery, and demonstrated that the amount and spatial pattern of bone formation was dependent on the delivery matrix. STATEMENT OF SIGNIFICANCE: Alginate hydrogel-based BMP-2 delivery has induced better spatiotemporal bone regeneration in animals, compared to clinically used collagen sponge, at lower BMP-2 doses. Lack of clear dose-response relationships for BMP-2 vis-à-vis bone regeneration has contributed to the use of higher doses clinically. We investigated the potential of the alginate system, with comparatively favorable BMP-2 release-kinetics, to reduce heterotopic ossification and promote bone regeneration, when used with a high BMP-2 dose. While defect mineralization improved with alginate hydrogel, the initial high-release phase and likely early tissue exposure to BMP-2 appeared sufficient to induce heterotopic ossification. The characterization presented here should provide the framework for future evaluations of strategies to optimize bone formation and minimize adverse effects of high dose BMP-2 therapy.