Intra-articular delivery of micronized dehydrated human amnion/chorion membrane reduces degenerative changes after onset of post-traumatic osteoarthritis.

Background: Micronized dehydrated human amnion/chorion membrane (mdHACM) has reduced short term post-traumatic osteoarthritis (PTOA) progression in rats when delivered 24 h after medial meniscal transection (MMT) and is being investigated for clinical use as a disease modifying therapy. Much remains to be assessed, including its potential for longer-term therapeutic benefit and treatment effects after onset of joint degeneration. Objectives: Characterize longer-term effects of acute treatment with mdHACM and determine whether treatment administered to joints with established PTOA could slow or reverse degeneration. Hypotheses: Acute treatment effects will be sustained for 6 weeks, and delivery of mdHACM after onset of joint degeneration will attenuate structural osteoarthritic changes. Methods: Rats underwent MMT or sham surgery (left leg). mdHACM was delivered intra-articularly 24 h or 3 weeks post-surgery (n = 5-7 per group). Six weeks post-surgery, animals were euthanized and left tibiae scanned using equilibrium partitioning of an ionic contrast agent microcomputed tomography (EPIC-µCT) to structurally quantify joint degeneration. Histology was performed to examine tibial plateau cartilage. Results: Quantitative 3D µCT showed that cartilage structural metrics (thickness, X-ray attenuation, surface roughness, exposed bone area) for delayed mdHACM treatment limbs were significantly improved over saline treatment and not significantly different from shams. Subchondral bone mineral density and thickness for the delayed treatment group were significantly improved over acute treated, and subchondral bone thickness was not significantly different from sham. Marginal osteophyte degenerative changes were decreased with delayed mdHACM treatment compared to saline. Acute treatment (24 h post-surgery) did not reduce longer-term joint tissue degeneration compared to saline. Histology supported µCT findings and further revealed that while delayed treatment reduced cartilage damage, chondrocytes displayed qualitatively different morphologies and density compared to sham. Conclusion: This study provides insight into effects of intra-articular delivery timing relative to PTOA progression and the duration of therapeutic benefit of mdHACM. Results suggest that mdHACM injection into already osteoarthritic joints can improve joint health, but a single, acute mdHACM injection post-injury does not prevent long term osteoarthritis associated with meniscal instability. Further work is needed to fully characterize the durability of therapeutic benefit in stable osteoarthritic joints and the effects of repeated injections.

Engineering of an Osteoinductive and Growth Factor-Free Injectable Bone-Like Microgel for Bone Regeneration.

Bone autografts remain the gold standard for bone grafting surgeries despite having increased donor site morbidity and limited availability. Bone morphogenetic protein-loaded grafts represent another successful commercial alternative. However, the therapeutic use of recombinant growth factors has been associated with significant adverse clinical outcomes. This highlights the need to develop biomaterials that closely approximate the structure and composition of bone autografts, which are inherently osteoinductive and biologically active with embedded living cells, without the need for added supplements. Here, injectable growth factor-free bone-like tissue constructs are developed, that closely approximate the cellular, structural, and chemical composition of bone autografts. It is demonstrated that these micro-constructs are inherently osteogenic, and demonstrate the ability to stimulate mineralized tissue formation and regenerate bone in critical-sized defects in-vivo. Furthermore, the mechanisms that allow human mesenchymal stem cells (hMSCs) to be highly osteogenic in these constructs, despite the lack of osteoinductive supplements, are assessed, whereby Yes activated protein (YAP) nuclear localization and adenosine signaling appear to regulate osteogenic cell differentiation. The findings represent a step toward a new class of minimally invasive, injectable, and inherently osteoinductive scaffolds, which are regenerative by virtue of their ability to mimic the tissue cellular and extracellular microenvironment, thus showing promise for clinical applications in regenerative engineering.

Leafy and weedy seadragon genomes connect genic and repetitive DNA features to the extravagant biology of syngnathid fishes.

Seadragons are a remarkable lineage of teleost fishes in the family Syngnathidae, renowned for having evolved male pregnancy. Comprising three known species, seadragons are widely recognized and admired for their fantastical body forms and coloration, and their specific habitat requirements have made them flagship representatives for marine conservation and natural history interests. Until recently, a gap has been the lack of significant genomic resources for seadragons. We have produced gene-annotated, chromosome-scale genome models for the leafy and weedy seadragon to advance investigations of evolutionary innovation and elaboration of morphological traits in seadragons as well as their pipefish and seahorse relatives. We identified several interesting features specific to seadragon genomes, including divergent noncoding regions near a developmental gene important for integumentary outgrowth, a high genome-wide density of repetitive DNA, and recent expansions of transposable elements and a vesicular trafficking gene family. Surprisingly, comparative analyses leveraging the seadragon genomes and additional syngnathid and outgroup genomes revealed striking, syngnathid-specific losses in the family of fibroblast growth factors (FGFs), which likely involve reorganization of highly conserved gene regulatory networks in ways that have not previously been documented in natural populations. The resources presented here serve as important tools for future evolutionary studies of developmental processes in syngnathids and hold value for conservation of the extravagant seadragons and their relatives.

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.

Reduced Size Profile of Amniotic Membrane Particles Decreases Osteoarthritis Therapeutic Efficacy.

Osteoarthritis (OA) is a widespread disease that continues to lack approved and efficacious treatments that modify disease progression. Micronized dehydrated human amnion/chorion membrane (µ-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. The objectives of this study were to characterize the particle size distributions of two µ-dHACM formulations and to investigate the influence of these distributions on the in vivo therapeutic efficacy of µ-dHACM. Male Lewis rats underwent medial meniscus transection (MMT) or sham surgery, and intra-articular injections of saline, µ-dHACM, or reduced particle size µ-dHACM (RPS µ-dHACM) were administered at 24 hours postsurgery (n = 9 per treatment group). After 3 weeks, the animals were euthanized, and left legs harvested for equilibrium partitioning of an ionic contrast agent microcomputed tomography and histological analysis. µ-dHACM and RPS µ-dHACM particles were fluorescently tagged and particle clearance was tracked in vivo for up to 42 days postsurgery. Protein elution from both formulations was quantified in vitro. Treatment with µ-HACM, but not RPS µ-dHACM, reduced lesion volume in the MMT model 3 weeks postsurgery. In contrast, RPS µ-dHACM increased cartilage surface roughness and osteophyte cartilage thickness and volume compared to saline treatment. There was no difference of in vivo fluorescently tagged particle clearance between the two µ-dHACM sizes. RPS µ-dHACM showed significantly greater protein elution in vitro over 21 days. Overall, delivery of RPS µ-dHACM did result in an increase of in vivo joint degeneration and in vitro protein elution compared to µ-dHACM, but did not result in differences in joint clearance in vivo. These results suggest that particle size and factor elution may be tailorable factors that are important to optimize for particulate amniotic membrane treatment to be an effective therapy for OA. Impact Statement Osteoarthritis (OA) is a widespread disease that continues to lack treatments that modify the progression of the disease. Micronized dehydrated human amnion/chorion membrane (µ-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. This work investigates the effects of particle size profile of the µ-dHACM particles and lays out the methods used in these studies. The results of this work will guide engineers in designing µ-dHACM treatments specifically and disease-modifying OA therapeutics generally, and it demonstrates the utility of novel therapeutic evaluation methods such as contrast-enhanced microcomputed tomography.

T1ρ, T2 mapping, and EPIC-µCT Imaging in a Canine Model of Knee Osteochondral Injury.

The dog is the most commonly used large animal model for the study of osteoarthritis. Optimizing methods for assessing cartilage health would prove useful in reducing the number of dogs needed for a valid study of osteoarthritis and cartilage repair. Twelve beagles had critical-sized osteochondral defects created in the medial femoral condyle of both knees. Eight dogs had T1ρ and T2 magnetic resonance imaging (MRI) performed approximately 6 months after defect creation. Following MRI evaluations, all 12 dogs were humanely euthanatized and cartilage samples were obtained from the medial and lateral femoral condyles, medial and lateral tibial plateaus, trochlear groove, and patella for proteoglycan and collagen quantification. Equilibrium partitioning of an ionic contrast (EPIC)-µCT was then performed followed by the histologic assessment of the knees. Correlations between T1ρ, T2, EPIC-µCT and proteoglycan, collagen, and histology scores were assessed using a multivariate analysis accounting for correlations from samples within the same knee and in the same dog. Pearson's correlation coefficients were calculated to assess the strength of significant relationships. Correlations between µCT values and biochemical or histologic assessment were weak to moderately strong (0.09-0.41; p < 0.0001-0.66). There was a weak correlation between the T2 values and cartilage proteoglycan (-0.32; p = 0.04). The correlation between T1ρ values and cartilage proteoglycan were moderately strong (-0.38; p < 0.05) while the strongest correlation was between the T1ρ values and histological assessment of cartilage with a correlation coefficient of 0.58 (p < 0.0001). These data suggest that T1ρ shows promise for possible utility in the translational study of cartilage health and warrants further development in this species. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:368-377, 2020.

Effects of Surface Topography and Chemistry on Polyether-Ether-Ketone (PEEK) and Titanium Osseointegration.

STUDY DESIGN: An in vivo study examining the functional osseointegration of smooth, rough, and porous surface topographies presenting polyether-ether-ketone (PEEK) or titanium surface chemistry. OBJECTIVE: To investigate the effects of surface topography and surface chemistry on implant osseointegration. SUMMARY OF BACKGROUND DATA: Interbody fusion devices have been used for decades to facilitate fusion across the disc space, yet debate continues over their optimal surface topography and chemistry. Though both factors influence osseointegration, the relative effects of each are not fully understood. METHODS: Smooth, rough, and porous implants presenting either a PEEK or titanium surface chemistry were implanted into the proximal tibial metaphyses of 36 skeletally mature male Sprague Dawley rats. At 8 weeks, animals were euthanized and bone-implant interfaces were subjected to micro-computed tomography analysis (n = 12), histology (n = 4), and biomechanical pullout testing (n = 8) to assess functional osseointegration and implant fixation. RESULTS: Micro-computed tomography analysis demonstrated that bone ingrowth was 38.9 ±â€Š2.8% for porous PEEK and 30.7 ±â€Š3.3% for porous titanium (P = 0.07). No differences in fixation strength were detected between porous PEEK and porous titanium despite titanium surfaces exhibiting an overall increase in bone-implant contact compared with PEEK (P < 0.01). Porous surfaces exhibited increased fixation strength compared with smooth and rough surfaces regardless of surface chemistry (P < 0.05). Across all groups both surface topography and chemistry had a significant overall effect on fixation strength (P < 0.05), but topography accounted for 65.3% of the total variance (ω = 0.65), whereas surface chemistry accounted for 5.9% (ω = 0.06). CONCLUSIONS: The effect of surface topography (specifically porosity) dominated the effect of surface chemistry in this study and could lead to further improvements in orthopedic device design. The poor osseointegration of existing smooth PEEK implants may be linked more to their smooth surface topography rather than their material composition. LEVEL OF EVIDENCE: N/A.

Porous PEEK improves the bone-implant interface compared to plasma-sprayed titanium coating on PEEK.

Polyether-ether-ketone (PEEK) is one of the most common materials used for load-bearing orthopaedic devices due to its radiolucency and favorable mechanical properties. However, current smooth-surfaced PEEK implants can lead to fibrous encapsulation and poor osseointegration. This study compared the in vitro and in vivo bone response to two smooth PEEK alternatives: porous PEEK and plasma-sprayed titanium coatings on PEEK. MC3T3 cells were grown on smooth PEEK, porous PEEK, and Ti-coated PEEK for 14 days and assayed for calcium content, osteocalcin, VEGF and ALP activity. Osseointegration was investigated by implanting cylindrical implants into the proximal tibiae of male Sprague Dawley rats for 8 weeks. Bone-implant interfaces were evaluated using µCT, histology and pullout testing. Cells on porous PEEK surfaces produced more calcium, osteocalcin, and VEGF than smooth PEEK and Ti-coated PEEK groups. Bone ingrowth into porous PEEK surfaces was comparable to previously reported porous materials and correlated well between µCT and histology analysis. Porous PEEK implants exhibited greater pullout force, stiffness and energy-to-failure compared to smooth PEEK and Ti-coated PEEK, despite Ti-coated PEEK exhibiting a high degree of bone-implant contact. These results are attributed to increased mechanical interlocking of bone with the porous PEEK implant surface. Overall, porous PEEK was associated with improved osteogenic differentiation in vitro and greater implant fixation in vivo compared to smooth PEEK and Ti-coated PEEK. These results suggest that not all PEEK implants inherently generate a fibrous response and that topography has a central role in determining implant osseointegration.

Biological evaluation and finite-element modeling of porous poly(para-phenylene) for orthopaedic implants.

Poly(para-phenylene) (PPP) is a novel aromatic polymer with higher strength and stiffness than polyetheretherketone (PEEK), the gold standard material for polymeric load-bearing orthopaedic implants. The amorphous structure of PPP makes it relatively straightforward to manufacture different architectures, while maintaining mechanical properties. PPP is promising as a potential orthopaedic material; however, the biocompatibility and osseointegration have not been well investigated. The objective of this study was to evaluate biological and mechanical behavior of PPP, with or without porosity, in comparison to PEEK. We examined four specific constructs: 1) solid PPP, 2) solid PEEK, 3) porous PPP and 4) porous PEEK. Pre-osteoblasts (MC3T3) exhibited similar cell proliferation among the materials. Osteogenic potential was significantly increased in the porous PPP scaffold as assessed by ALP activity and calcium mineralization. In vivo osseointegration was assessed by implanting the cylindrical materials into a defect in the metaphysis region of rat tibiae. Significantly more mineral ingrowth was observed in both porous scaffolds compared to the solid scaffolds, and porous PPP had a further increase compared to porous PEEK. Additionally, porous PPP implants showed bone formation throughout the porous structure when observed via histology. A computational simulation of mechanical push-out strength showed approximately 50% higher interfacial strength in the porous PPP implants compared to the porous PEEK implants and similar stress dissipation. These data demonstrate the potential utility of PPP for orthopaedic applications and show improved osseointegration when compared to the currently available polymeric material. STATEMENT OF SIGNIFICANCE: PEEK has been widely used in orthopaedic surgery; however, the ability to utilize PEEK for advanced fabrication methods, such as 3D printing and tailored porosity, remain challenging. We present a promising new orthopaedic biomaterial, Poly(para-phenylene) (PPP), which is a novel class of aromatic polymers with higher strength and stiffness than polyetheretherketone (PEEK). PPP has exceptional mechanical strength and stiffness due to its repeating aromatic rings that provide strong anti-rotational biaryl bonds. Furthermore, PPP has an amorphous structure making it relatively easier to manufacture (via molding or solvent-casting techniques) into different geometries with and without porosity. This ability to manufacture different architectures and use different processes while maintaining mechanical properties makes PPP a very promising potential orthopaedic biomaterial which may allow for closer matching of mechanical properties between the host bone tissue while also allowing for enhanced osseointegration. In this manuscript, we look at the potential of porous and solid PPP in comparison to PEEK. We measured the mechanical properties of PPP and PEEK scaffolds, tested these scaffolds in vitro for osteocompatibility with MC3T3 cells, and then tested the osseointegration and subsequent functional integration in vivo in a metaphyseal drill hole model in rat tibia. We found that PPP permits cell adhesion, growth, and mineralization in vitro. In vivo it was found that porous PPP significantly enhanced mineralization into the construct and increased the mechanical strength required to push out the scaffold in comparison to PEEK. This is the first study to investigate the performance of PPP as an orthopaedic biomaterial in vivo. PPP is an attractive material for orthopaedic implants due to the ease of manufacturing and superior mechanical strength.

The effect of contouring on fatigue resistance of three types of fracture fixation plates.

BACKGROUND: Metallic reconstruction plates used for fracture stabilization typically require intraoperative contouring for patient-specific anatomical fit. Despite this, characterization of plate mechanical properties after contouring has previously been limited. The objective of this study was to assess whether contouring affects fatigue resistance for three types of Stryker seven-hole stainless steel (SS) 316LVM fracture fixation plates. The hypothesis was that for each plate type, more contouring repetitions would result in lower fatigue resistance. METHODS: Plates were contoured using a bench-top plate bender to ±20° either 0×, 3×, 6×, or 9× (n = 5 per group) and tested in the straight configuration. Cyclic four-point bending was applied in an incremental stepwise staircase approach (one step = 100,000 cycles, 10 Hz) until failure (defined as brittle fracture or plastic deformation of 10° permanent bend). Moment-cycle product (MCP) was computed as the summation of maximum moment × number of cycles and used as the primary measure of fatigue resistance. RESULTS: No significant differences in fatigue resistance were detected between contouring groups for Basic Fragment Set (BFS) Reconstruction Plates. Significantly lower fatigue resistance was measured for 9× contoured Matta Pelvic System (MPS) Straight Plates compared to 0× contoured plates (p = 0.023). MPS Flex Plates contoured 3× had greater fatigue resistance than 0× contoured (p = 0.031) and 9× contoured plates (p = 0.032). CONCLUSIONS: This work provides fatigue resistance-based evidence that clinicians should avoid high repetitions of contouring for MPS Straight Plates. Meanwhile, BFS Reconstruction Plates and MPS Flex Plates are not negatively affected by contouring. These results allow for improved intraoperative decisions about using or discarding plates after multiple contouring repetitions.

Influence of structural load-bearing scaffolds on mechanical load- and BMP-2-mediated bone regeneration.

A common design constraint in functional tissue engineering is that scaffolds intended for use in load-bearing sites possess similar mechanical properties to the replaced tissue. Here, we tested the hypothesis that in vivo loading would enhance bone morphogenetic protein-2 (BMP-2)-mediated bone regeneration in the presence of a load-bearing PLDL scaffold, whose pores and central core were filled with BMP-2-releasing alginate hydrogel. First, we evaluated the effects of in vivo mechanical loading on bone regeneration in the structural scaffolds. Second, we compared scaffold-mediated bone regeneration, independent of mechanical loading, with alginate hydrogel constructs, without the structural scaffold, that have been shown previously to facilitate in vivo mechanical stimulation of bone formation. Contrary to our hypothesis, mechanical loading had no effect on bone formation, distribution, or biomechanical properties in structural scaffolds. Independent of loading, the structural scaffolds reduced bone formation compared to non-structural alginate, particularly in regions in which the scaffold was concentrated, resulting in impaired functional regeneration. This is attributable to a combination of stress shielding by the scaffold and inhibition of cellular infiltration and tissue ingrowth. Collectively, these data question the necessity of scaffold similarity to mature tissue at the time of implantation and emphasize development of an environment conducive to cellular activation of matrix production and ultimate functional regeneration.

EPIC-µCT imaging of articular cartilage.

Characterization of articular cartilage morphology and composition using microcomputed tomography (microCT) techniques requires the use of contrast agents to enhance X-ray attenuation of the tissue. This chapter describes the use of an anionic iodinated contrast agent at equilibrium with articular cartilage. In this technique, negatively charged contrast agent molecules distribute themselves inversely with respect to the negatively charged proteoglycans (PGs) within the cartilage tissue (Palmer et al. Proc Natl Acad Sci U S A 103:19255-19260, 2006). This relationship allows for assessment of cartilage degradation, as areas of high X-ray attenuation have been shown to correspond to areas of depleted PGs (Palmer et al. Proc Natl Acad Sci U S A 103:19255-19260, 2006; Xie et al. Osteoarthritis Cartilage 18:65-72, 2010).

High-strength, surface-porous polyether-ether-ketone for load-bearing orthopedic implants.

Despite its widespread clinical use in load-bearing orthopedic implants, polyether-ether-ketone (PEEK) is often associated with poor osseointegration. In this study, a surface-porous PEEK material (PEEK-SP) was created using a melt extrusion technique. The porous layer was 399.6±63.3 µm thick and possessed a mean pore size of 279.9±31.6 µm, strut spacing of 186.8±55.5 µm, porosity of 67.3±3.1% and interconnectivity of 99.9±0.1%. Monotonic tensile tests showed that PEEK-SP preserved 73.9% of the strength (71.06±2.17 MPa) and 73.4% of the elastic modulus (2.45±0.31 GPa) of as-received, injection-molded PEEK. PEEK-SP further demonstrated a fatigue strength of 60.0 MPa at one million cycles, preserving 73.4% of the fatigue resistance of injection-molded PEEK. Interfacial shear testing showed the pore layer shear strength to be 23.96±2.26 MPa. An osseointegration model in the rat revealed substantial bone formation within the pore layer at 6 and 12 weeks via microcomputed tomography and histological evaluation. Ingrown bone was more closely apposed to the pore wall and fibrous tissue growth was reduced in PEEK-SP when compared to non-porous PEEK controls. These results indicate that PEEK-SP could provide improved osseointegration while maintaining the structural integrity necessary for load-bearing orthopedic applications.

Intra-articular injection of micronized dehydrated human amnion/chorion membrane attenuates osteoarthritis development.

INTRODUCTION: Micronized dehydrated human amnion/chorion membrane (µ-dHACM) is derived from donated human placentae and has anti-inflammatory, low immunogenic and anti-fibrotic properties. The objective of this study was to quantitatively assess the efficacy of µ-dHACM as a disease modifying intervention in a rat model of osteoarthritis (OA). It was hypothesized that intra-articular injection of µ-dHACM would attenuate OA progression. METHODS: Lewis rats underwent medial meniscal transection (MMT) surgery to induce OA. Twenty four hours post-surgery, µ-dHACM or saline was injected intra-articularly into the rat joint. Naïve rats also received µ-dHACM injections. Microstructural changes in the tibial articular cartilage were assessed using equilibrium partitioning of an ionic contrast agent (EPIC-µCT) at 21 days post-surgery. The joint was also evaluated histologically and synovial fluid was analyzed for inflammatory markers at 3 and 21 days post-surgery. RESULTS: There was no measured baseline effect of µ-dHACM on cartilage in naïve animals. Histological staining of treated joints showed presence of µ-dHACM in the synovium along with local hypercellularity at 3 and 21 days post-surgery. In MMT animals, development of cartilage lesions at 21 days was prevented and number of partial erosions was significantly reduced by treatment with µ-dHACM. EPIC-µCT analysis quantitatively showed that µ-dHACM reduced proteoglycan loss in MMT animals. CONCLUSIONS: µ-dHACM is rapidly sequestered in the synovial membrane following intra-articular injection and attenuates cartilage degradation in a rat OA model. These data suggest that intra-articular delivery of µ-dHACM may have a therapeutic effect on OA development.

Particulated Juvenile Articular Cartilage Implantation in the Knee: A 3-Year EPIC-µCT and Histological Examination.

OBJECTIVE: The goal of this report is to describe the outcome of sequential particulated cartilage allograft and autologous osteochondral transfer treatments for an osteochondral lesion of the medial femoral condyle. METHODS: A 44-year-old woman was treated with a particulated juvenile articular cartilage allograft (DeNovo NT) for a chondral lesion of the knee. As a result of continued pain, she had 2 further surgeries, including an autologous osteochondral transfer system procedure and finally a unicondylar knee arthroplasty. At the final procedure, the areas of the allograft and autograft tissue were biopsied for histological evaluation. The quality of the residual cartilage tissue was assessed first by equilibrium partitioning of an ionic contrast agent via micro-computed tomography (EPIC-µCT), and then by hematoxylin and eosin, Safranin O staining, and polarized light microscopy. RESULTS: Despite showing good healing at 7 months postsurgery by MRI, at 28 months post DeNovo NT tissue implantation the excised cartilage tissue was heterogeneous, with some regions of hyaline-like cartilage and some regions of fibrocartilage. The later mosaicplasty may have helped maintain hyaline-like cartilage of the DeNovo NT tissue in its vicinity. CONCLUSION: This case report describes the cartilage repair tissue produced by DeNovo NT implantation and compares it with autologous osteochondral plug tissue.

Quantitative imaging of cartilage and bone morphology, reactive oxygen species, and vascularization in a rodent model of osteoarthritis.

OBJECTIVE: To assess temporal changes in cartilage and bone morphology, reactive oxygen species (ROS), and vascularization in rats with monosodium iodoacetate (MIA)-induced osteoarthritis (OA), using advanced imaging methodologies. METHODS: Right knees of 8-week-old male Wistar rats were injected with 1 mg MIA in 50 µl saline and left knees were injected with 50 µl saline as controls. After 1, 2, and 3 weeks (n = 5 at each time point), changes in cartilage morphology and composition were quantified using equilibrium partitioning of an ionic contrast agent microfocal computed tomography (µCT), and changes in subchondral and trabecular bone were assessed by standard µCT. ROS were characterized by in vivo fluorescence imaging at 1, 11, and 21 days (n = 5 at each time point). Three weeks following fluorescence imaging, alterations in knee joint vascularity were quantified with µCT after perfusion of a vascular contrast agent. RESULTS: Femoral cartilage volume, thickness, and proteoglycan content were significantly decreased in MIA-injected knees compared with control knees, accompanied by loss of trabecular bone and erosion of subchondral bone surface. ROS quantities were significantly increased 1 day after MIA injection and subsequently decreased gradually, having returned to normal by 21 days. Vascularity in whole knees and distal femora was significantly increased at 21 days after MIA injection. CONCLUSION: Contrast-enhanced µCT and fluorescence imaging were combined to characterize articular cartilage, subchondral bone, vascularization, and ROS, providing unprecedented 3-dimensional joint imaging and quantification in multiple tissues during OA progression. These advanced imaging techniques have the potential to become standardized methods for comprehensive evaluation of articular joint degeneration and evaluation of therapeutic efficacy.

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.

Suprachoroidal drug delivery to the back of the eye using hollow microneedles.

PURPOSE: In this work, we tested the hypothesis that microneedles provide a minimally invasive method to inject particles into the suprachoroidal space for drug delivery to the back of the eye. METHODS: A single, hollow microneedle was inserted into the sclera, and infused nanoparticle and microparticle suspensions into the suprachoroidal space. Experiments were performed on whole rabbit, pig, and human eyes ex vivo. Particle delivery was imaged using brightfield and fluorescence microscopy as well as microcomputed tomography. RESULTS: Microneedles were shown to deliver sulforhodamine B as well as nanoparticle and microparticle suspensions into the suprachoroidal space of rabbit, pig, and human eyes. Volumes up to 35 µL were administered consistently. Optimization of the delivery device parameters showed that microneedle length, pressure, and particle size played an important role in determining successful delivery into the suprachoroidal space. Needle lengths of 800-1,000 µm and applied pressures of 250-300 kPa provided most reliable delivery. CONCLUSIONS: Microneedles were shown for the first time to deliver nanoparticle and microparticle suspensions into the suprachoroidal space of rabbit, pig and human eyes. This shows that microneedles may provide a minimally invasive method for controlled drug delivery to the back of the eye.

Hyaluronic acid hydrogels with controlled degradation properties for oriented bone regeneration.

Non-healing fractures can result from trauma, disease, or age-related bone loss. While many treatments focus on restoring bone volume, few try to recapitulate bone organization. However, the native architecture of bone is optimized to provide its necessary mechanical properties. Hyaluronic acid (HA) hydrogel scaffold systems with tunable degradation properties were developed for the controlled delivery of osteoinductive and angiogenic growth factors, thus affecting the quantity and quality of regenerated tissue. HA hydrogels were designed to degrade at fast, intermediate, and slow rates due to hydrolysis and further provided controlled release of cationic proteins due to electrostatic interactions. Scaffolds delivering bone morphogenetic protein-2 (BMP-2) were evaluated in a rat calvarial bone critical size defect model. BMP-2 delivery from the HA hydrogels had a clear osteoinductive effect in vivo and, for all hydrogel types, BMP-2 delivery resulted in significant mineralization compared to control hydrogels. The temporal progression of this effect could be modulated by altering the degradation rate of the scaffold. All three degradation rates tested resulted in similar amounts of mineral formation at the latest (six week) time point examined. Interestingly, however, the fastest and slowest degrading scaffolds seemed to result in more organized bone than the intermediate degrading scaffold, which was designed to degrade in 6-8 weeks to match the healing time. Additionally, healing could be enhanced by co-delivery of vascular endothelial growth factor along with BMP-2.

Rescue of impaired fracture healing in COX-2-/- mice via activation of prostaglandin E2 receptor subtype 4.

Although the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracture-healing model, COX-2(-/-) mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2(-/-) mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2(-/-) callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2(-/-) mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 long-term limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9 expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9 expression in the COX-2(-/-) fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2(-/-) mice.