Flow Preconditioning of Endothelial Cells on Collagen-Immobilized Silicone Fibers Enhances Cell Retention and Antithrombotic Function.

Stability and antithrombotic functionality of endothelial cells on silicone hollow fibers (SiHFs) are critical in the development of biohybrid artificial lungs. Here we aimed to enhance endothelial cell retention and anti-thrombotic function by low (12 dyn/cm2 , 24 h) fluid shear stress ("flow") preconditioning of endothelial cells seeded on collagen-immobilized SiHFs. The response of endothelial cells without preconditioning (48 h static culture) and with preconditioning (24 h static culture followed by 24 h flow preconditioning) on hollow fibers to high fluid shear stress (30 dyn/cm2 , 1 h) was assessed in a parallel-plate flow chamber. Finite element (FE) modeling was used to simulate shear stress within the flow chamber. We found that collagen immobilization on hollow fibers using carbodiimide bonds provided sufficient stability to high shear stress. Flow preconditioning for 24 h before treatment with high shear stress for 1 h on collagen-immobilized hollow fibers increased cell retention (1.3-fold). The FE model showed that cell flattening due to flow preconditioning reduced maximum shear stress on cells by 32%. Flow preconditioning prior to exposure to high fluid shear stress enhanced the production of nitric oxide (1.3-fold) and prostaglandin I2 (1.2-fold). In conclusion, flow preconditioning of endothelial cells on collagen-immobilized SiHFs enhanced cell retention and antithrombotic function, which could significantly improve current biohybrid artificial lungs.

Applying shear stress to endothelial cells in a new perfusion chamber: hydrodynamic analysis.

Perfusion bioreactors have been proved to be an impartible part of vascular tissue engineering due to its broad range of applications as a means to distribute nutrients within porous scaffold along with providing appropriate physical and mechanical stimuli. To better understand the mechanical phenomena inside a bioreactor, computational fluid dynamics (CFD) was adopted followed by a validation technique. The fluid dynamics of the media inside the bioreactor was modeled using the Navier-Stokes equation for incompressible fluids while convection through the scaffold was described by Brinkman's extension of Darcy's law for porous media. Flow within the reactor determined the orientation of endothelial cells on the scaffold. To validate flow patterns, streamlines and shear stresses, colorimetry technique was used following attained results from CFD. Our bioreactor was modeled to simulate the optimum condition and flow patterns over scaffold to culture ECs for in vitro experimentation. In such experiments, cells were attached firmly without significant detachment and more noticeably elongation process was triggered even shortly after start up.

Dual-contrast micro-CT enables cartilage lesion detection and tissue condition evaluation ex vivo.

BACKGROUND: Post-traumatic osteoarthritis is a frequent joint disease in the horse. Currently, equine medicine lacks effective methods to diagnose the severity of chondral defects after an injury. OBJECTIVES: To investigate the capability of dual-contrast-enhanced computed tomography (dual-CECT) for detection of chondral lesions and evaluation of the severity of articular cartilage degeneration in the equine carpus ex vivo. STUDY DESIGN: Pre-clinical experimental study. METHODS: In nine Shetland ponies, blunt and sharp grooves were randomly created (in vivo) in the cartilage of radiocarpal and middle carpal joints. The contralateral joint served as control. The ponies were subjected to an 8-week exercise protocol and euthanised 39 weeks after surgery. CECT scanning (ex vivo) of the joints was performed using a micro-CT scanner 1 hour after an intra-articular injection of a dual-contrast agent. The dual-contrast agent consisted of ioxaglate (negatively charged, q = -1) and bismuth nanoparticles (BiNPs, q = 0, diameter ≈ 0.2 µm). CECT results were compared to histological cartilage proteoglycan content maps acquired using digital densitometry. RESULTS: BiNPs enabled prolonged visual detection of both groove types as they are too large to diffuse into the cartilage. Furthermore, proportional ioxaglate diffusion inside the tissue allowed differentiation between the lesion and ungrooved articular cartilage (3 mm from the lesion and contralateral joint). The mean ioxaglate partition in the lesion was 19 percentage points higher (P < 0.001) when compared with the contralateral joint. The digital densitometry and the dual-contrast CECT findings showed good subjective visual agreement. MAIN LIMITATIONS: Ex vivo study protocol and a low number of investigated joints. CONCLUSIONS: The dual-CECT methodology, used in this study for the first time to image whole equine joints, is capable of effective lesion detection and simultaneous evaluation of the condition of the articular cartilage.

Effects of human articular cartilage constituents on simultaneous diffusion of cationic and nonionic contrast agents.

Contrast-enhanced computed tomography is an emerging diagnostic technique for osteoarthritis. However, the effects of increased water content, as well as decreased collagen and proteoglycan concentrations due to cartilage degeneration, on the diffusion of cationic and nonionic agents, are not fully understood. We hypothesize that for a cationic agent, these variations increase the diffusion rate while decreasing partition, whereas, for a nonionic agent, these changes increase both the rate of diffusion and partition. Thus, we examine the diffusion of cationic and nonionic contrast agents within degraded tissue in time- and depth-dependent manners. Osteochondral plugs (N = 15, d = 8 mm) were extracted from human cadaver knee joints, immersed in a mixture of cationic CA4+ and nonionic gadoteridol contrast agents, and imaged at multiple time-points, using the dual-contrast method. Water content, and collagen and proteoglycan concentrations were determined using lyophilization, infrared spectroscopy, and digital densitometry, respectively. Superficial to mid (0%-60% depth) cartilage CA4+ partitions correlated with water content (R < -0.521, P < .05), whereas in deeper (40%-100%) cartilage, CA4+ correlated only with proteoglycans (R > 0.671, P < .01). Gadoteridol partition correlated inversely with collagen concentration (0%-100%, R < -0.514, P < .05). Cartilage degeneration substantially increased the time for CA4+ compared with healthy tissue (248 ± 171 vs 175 ± 95 minute) to reach the bone-cartilage interface, whereas for gadoteridol the time (111 ± 63 vs 179 ± 163 minute) decreased. The work clarifies the diffusion mechanisms of two different contrast agents and presents depth and time-dependent effects resulting from articular cartilage constituents. The results will inform the development of new contrast agents and optimal timing between agent administration and joint imaging.

Topographic features of nano-pores within the osteochondral interface and their effects on transport properties -a 3D imaging and modeling study.

Recent insights suggest that the osteochondral interface plays a central role in maintaining healthy articulating joints. Uncovering the underlying transport mechanisms is key to the understanding of the cross-talk between articular cartilage and subchondral bone. Here, we describe the mechanisms that facilitate transport at the osteochondral interface. Using scanning electron microscopy (SEM), we found a continuous transition of mineralization architecture from the non-calcified cartilage towards the calcified cartilage. This refurbishes the classical picture of the so-called tidemark; a well-defined discontinuity at the osteochondral interface. Using focused-ion-beam SEM (FIB-SEM) on one osteochondral plug derived from a human cadaveric knee, we elucidated that the pore structure gradually varies from the calcified cartilage towards the subchondral bone plate. We identified nano-pores with radius of 10.71 ± 6.45 nm in calcified cartilage to 39.1 ± 26.17 nm in the subchondral bone plate. The extracted pore sizes were used to construct 3D pore-scale numerical models to explore the effect of pore sizes and connectivity among different pores. Results indicated that connectivity of nano-pores in calcified cartilage is highly compromised compared to the subchondral bone plate. Flow simulations showed a permeability decrease by about 2000-fold and solute transport simulations using a tracer (iodixanol, 1.5 kDa with a free diffusivity of 2.5 × 10-10 m2/s) showed diffusivity decrease by a factor of 1.5. Taken together, architecture of the nano-pores and the complex mineralization pattern in the osteochondral interface considerably impacts the cross-talk between cartilage and bone.

Structural, compositional, and functional effects of blunt and sharp cartilage damage on the joint: A 9-month equine groove model study.

This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and their effect on joint homeostasis and function of the equine carpus. In nine adult Shetland ponies, the cartilage in the radiocarpal and middle carpal joint of one front limb was grooved (blunt or sharp randomized). The ponies were subjected to an 8-week exercise protocol and euthanized at 39 weeks. Structural and compositional alterations in joint tissues were evaluated in vivo using serial radiographs, synovial biopsies, and synovial fluid samples. Joint function was monitored by quantitative gait analysis. Macroscopic, microscopic, and biomechanical evaluation of the cartilage and assessment of subchondral bone parameters were performed ex vivo. Grooved cartilage showed higher OARSI microscopy scores than the contra-lateral sham-operated controls (p < 0.0001). Blunt-grooved cartilage scored higher than sharp-grooved cartilage (p = 0.007) and fixed charge density around these grooves was lower (p = 0.006). Equilibrium and instantaneous moduli trended lower in grooved cartilage than their controls (significant for radiocarpal joints). Changes in other tissues included a threefold to sevenfold change in interleukin-6 expression in synovium from grooved joints at week 23 (p = 0.042) and an increased CPII/C2C ratio in synovial fluid extracted from blunt-grooved joints at week 35 (p = 0.010). Gait analysis outcome revealed mild, gradually increasing lameness. In conclusion, blunt and, to a lesser extent, sharp grooves in combination with a period of moderate exercise, lead to mild degeneration in equine carpal cartilage over a 9-month period, but the effect on overall joint health remains limited.

3D-printed saw guides for lower arm osteotomy, a comparison between a synthetic CT and CT-based workflow.

BACKGROUND: Three-dimensional (3D)-printed saw guides are frequently used to optimize osteotomy results and are usually designed based on computed tomography (CT), despite the radiation burden, as radiation-less alternatives like magnetic resonance imaging (MRI) have inferior bone visualization capabilities. This study investigated the usability of MR-based synthetic-CT (sCT), a novel radiation-less bone visualization technique for 3D planning and design of patient-specific saw guides. METHODS: Eight human cadaveric lower arms (mean age: 78y) received MRI and CT scans as well as high-resolution micro-CT. From the MRI scans, sCT were generated using a conditional generative adversarial network. Digital 3D bone surface models based on the sCT and general CT were compared to the surface model from the micro-CT that was used as ground truth for image resolution. From both the sCT and CT digital bone models saw guides were designed and 3D-printed in nylon for one proximal and one distal bone position for each radius and ulna. Six blinded observers placed these saw guides as accurately as possible on dissected bones. The position of each guide was assessed by optical 3D-scanning of each bone with positioned saw guide and compared to the preplanning. Eight placement errors were evaluated: three translational errors (along each axis), three rotational errors (around each axis), a total translation (∆T) and a total rotation error (∆R). RESULTS: Surface models derived from micro-CT were on average smaller than sCT and CT-based models with average differences of 0.27 ± 0.30 mm for sCT and 0.24 ± 0.12 mm for CT. No statistically significant positioning differences on the bones were found between sCT- and CT-based saw guides for any axis specific translational or rotational errors nor between the ∆T (p = .284) and ∆R (p = .216). On Bland-Altman plots, the ∆T and ∆R limits of agreement (LoA) were within the inter-observer variability LoA. CONCLUSIONS: This research showed a similar error for sCT and CT digital surface models when comparing to ground truth micro-CT models. Additionally, the saw guide study showed equivalent CT- and sCT-based saw guide placement errors. Therefore, MRI-based synthetic CT is a promising radiation-less alternative to CT for the creation of patient-specific osteotomy surgical saw guides.

Orthotopic Bone Regeneration within 3D Printed Bioceramic Scaffolds with Region-Dependent Porosity Gradients in an Equine Model.

The clinical translation of three-dimensionally printed bioceramic scaffolds with tailored architectures holds great promise toward the regeneration of bone to heal critical-size defects. Herein, the long-term in vivo performance of printed hydrogel-ceramic composites made of methacrylated-oligocaprolactone-poloxamer and low-temperature self-setting calcium-phosphates is assessed in a large animal model. Scaffolds printed with different internal architectures, displaying either a designed porosity gradient or a constant pore distribution, are implanted in equine tuber coxae critical size defects. Bone ingrowth is challenged and facilitated only from one direction via encasing the bioceramic in a polycaprolactone shell. After 7 months, total new bone volume and scaffold degradation are significantly greater in structures with constant porosity. Interestingly, gradient scaffolds show lower extent of remodeling and regeneration even in areas having the same porosity as the constant scaffolds. Low regeneration in distal regions from the interface with native bone impairs ossification in proximal regions of the construct, suggesting that anisotropic architectures modulate the cross-talk between distant cells within critical-size defects. The study provides key information on how engineered architectural patterns impact osteoregeneration in vivo, and also indicates the equine tuber coxae as promising orthotopic model for studying materials stimulating bone formation.

Dual contrast in computed tomography allows earlier characterization of articular cartilage over single contrast.

Cationic computed tomography contrast agents are more sensitive for detecting cartilage degeneration than anionic or non-ionic agents. However, osteoarthritis-related loss of proteoglycans and increase in water content contrarily affect the diffusion of cationic contrast agents, limiting their sensitivity. The quantitative dual-energy computed tomography technique allows the simultaneous determination of the partitions of iodine-based cationic (CA4+) and gadolinium-based non-ionic (gadoteridol) agents in cartilage at diffusion equilibrium. Normalizing the cationic agent partition at diffusion equilibrium with that of the non-ionic agent improves diagnostic sensitivity. We hypothesize that this sensitivity improvement is also prominent during early diffusion time points and that the technique is applicable during contrast agent diffusion. To investigate the validity of this hypothesis, osteochondral plugs (d = 8 mm, N = 33), extracted from human cadaver (n = 4) knee joints, were immersed in a contrast agent bath (a mixture of CA4+ and gadoteridol) and imaged using the technique at multiple time points until diffusion equilibrium. Biomechanical testing and histological analysis were conducted for reference. Quantitative dual-energy computed tomography technique enabled earlier determination of cartilage proteoglycan content over single contrast. The correlation coefficient between human articular cartilage proteoglycan content and CA4+ partition increased with the contrast agent diffusion time. Gadoteridol normalized CA4+ partition correlated significantly (P < .05) with Mankin score at all time points and with proteoglycan content after 4 hours. The technique is applicable during diffusion, and normalization with gadoteridol partition improves the sensitivity of the CA4+ contrast agent.

The Osteoinductive Effect of Controlled Bone Morphogenic Protein 2 Release Is Location Dependent.

IMPACT STATEMENT: The main challenge in bone morphogenic protein 2 (BMP-2)-based application lies in finding strategies to prolong its biologic activity as it has a short biological half-life. The present study uses a phosphate-modified oligo[(polyethylene glycol) fumarate] hydrogel that can be tuned to achieve differential release profiles of biologically active BMP-2 release. We demonstrate that this platform outperforms Infuse®, currently used in the clinic and that the osteoinductive effect of BMP-2 is location dependent. Altogether, this study stresses the importance of evaluating efficacy of bone tissue engineering strategies at an orthotopic location rather than subcutaneously. Even more so, it emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue engineering.

Comparison of polyetheretherketone versus silicon nitride intervertebral spinal spacers in a caprine model.

Polyetheretherketone (PEEK) is commonly used as a spinal spacer for intervertebral fusion surgery. Unfortunately, PEEK is bioinert and does not effectively osseointegrate into living bone. In contrast, comparable spacers made of silicon nitride (Si3 N4 ) possess a surface nanostructure and chemistry that encourage appositional bone healing. This observational study was designed to compare the outcomes of these two biomaterials when implanted as spacers in an adult caprine model. Lumbar interbody fusion surgeries were performed at two adjacent levels in eight adult goats using implants of PEEK and Si3 N4 . At six-months after surgery, the operative and adjacent spinal segments were extracted and measured for bone fusion, bone volume, bone-implant contact (BIC) and soft-tissue implant contact (SIC) ratios, and biodynamic stability. The null hypothesis was that no differences in these parameters would be apparent between the two groups. Fusion was observed in seven of eight implants in each group with greater bone formation in the Si3 N4 group (52.6%) versus PEEK (27.9%; p = 0.2). There were no significant differences in BIC ratios between PEEK and Si3 N4 , and the biodynamic stability of the two groups was also comparable. The results suggest that Si3 N4 spacers are not inferior to PEEK and they may be more effective in promoting arthrodesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 688-699, 2019.

Contrast enhanced computed tomography for real-time quantification of glycosaminoglycans in cartilage tissue engineered constructs.

Tissue engineering and regenerative medicine are two therapeutic strategies to treat, and to potentially cure, diseases affecting cartilaginous tissues, such as osteoarthritis and cartilage defects. Insights into the processes occurring during regeneration are essential to steer and inform development of the envisaged regenerative strategy, however tools are needed for longitudinal and quantitative monitoring of cartilage matrix components. In this study, we introduce a contrast-enhanced computed tomography (CECT)-based method using a cationic iodinated contrast agent (CA4+) for longitudinal quantification of glycosaminoglycans (GAG) in cartilage-engineered constructs. CA4+ concentration and scanning protocols were first optimized to ensure no cytotoxicity and a facile procedure with minimal radiation dose. Chondrocyte and mesenchymal stem cell pellets, containing different GAG content were generated and exposed to CA4+. The CA4+ content in the pellets, as determined by micro computed tomography, was plotted against GAG content, as measured by 1,9-dimethylmethylene blue analysis, and showed a high linear correlation. The established equation was used for longitudinal measurements of GAG content over 28 days of pellet culture. Importantly, this method did not adversely affect cell viability or chondrogenesis. Additionally, the CA4+ distribution accurately matched safranin-O staining on histological sections. Hence, we show proof-of-concept for the application of CECT, utilizing a positively charged contrast agent, for longitudinal and quantitative imaging of GAG distribution in cartilage tissue-engineered constructs. STATEMENT OF SIGNIFICANCE: Tissue engineering and regenerative medicine are promising therapeutic strategies for different joint pathologies such as cartilage defects or osteoarthritis. Currently, in vitro assessment on the quality and composition of the engineered cartilage mainly relies on destructive methods. Therefore, there is a need for the development of techniques that allow for longitudinal and quantitative imaging and monitoring of cartilage-engineered constructs. This work harnesses the electrostatic interactions between the negatively-charged glycosaminoglycans (GAGs) and a positively-charged contrast agent for longitudinal and non-destructive quantification of GAGs, providing valuable insight on GAG development and distribution in cartilage engineered constructs. Such technique can advance the development of regenerative strategies, not only by allowing continuous monitoring but also by serving as a pre-implantation screening tool.

Local controlled release of corticosteroids extends surgically induced joint instability by inhibiting tissue healing.

BACKGROUND AND PURPOSE: Corticosteroids are intra-articularly injected to relieve pain in joints with osteoarthritis (OA) or acute tissue damage such as ligament or tendon tears, despite its unverified contraindication in unstable joints. Biomaterial-based sustained delivery may prolong reduction of inflammatory pain, while avoiding harmful peak drug concentrations. EXPERIMENTAL APPROACH: The applicability of prolonged corticosteroid exposure was examined in a rat model of anterior cruciate ligament and medial meniscus transection (ACLT + pMMx) with ensuing degenerative changes. KEY RESULTS: Intra-articular injection of a bolus of the corticosteroid triamcinolone acetonide (TAA) resulted in enhanced joint instability in 50% of the joints, but neither instability-induced OA cartilage degeneration, synovitis, nor the OA-related bone phenotype was affected. However, biomaterial microsphere-based extended TAA release enhanced instability in 94% of the animals and induced dystrophic calcification and exacerbation of cartilage degeneration. In healthy joints, injection with TAA releasing microspheres had no effect at all. In vitro, TAA inhibited cell migration out of joint tissue explants, suggesting inhibited tissue healing in vivo as mechanisms for enhanced instability and subsequent cartilage degeneration. CONCLUSIONS AND IMPLICATIONS: We conclude that short-term TAA exposure has minor effects on surgically induced unstable joints, but its extended presence is detrimental by extending instability and associated joint degeneration through compromised healing. This supports a contraindication of prolonged corticosteroid exposure in tissue damage-associated joint instability, but not of brief exposure.

Unfocused shockwaves for osteoinduction in bone substitutes in rat cortical bone defects.

Bone substitutes are frequently used in clinical practice but often exhibit limited osteoinductivity. We hypothesized that unfocused shockwaves enhance the osteoinductivity of bone substitutes and improve osteointegration and angiogenesis. Three different bone substitutes, namely porous tricalcium phosphate, porous hydroxyapatite and porous titanium alloy, were implanted in a critical size (i.e. 6-mm) femoral defect in rats. The femora were treated twice with 1500 shockwaves at 2 and 4 weeks after surgery and compared with non-treated controls. The net volume of de novo bone in the defect was measured by microCT-scanning during 11-weeks follow-up. Bone ingrowth and angiogenesis in the bone substitutes was examined at 5 and 11 weeks using histology. It was shown that hydroxyapatite and titanium both had an increase of bone ingrowth with more bone in the shockwave group compared to the control group, whereas resorption was seen in tricalcium phosphate bone substitutes over time and this was insensitive to shockwave treatment. In conclusion, hydroxyapatite and titanium bone substitutes favour from shockwave treatment, whereas tricalcium phosphate does not. This study shows that osteoinduction and osteointegration of bone substitutes can be influenced with unfocused shockwave therapy, but among other factors depend on the type of bone substitute, likely reflecting its mechanical and biological properties.

Early Signs of Bone and Cartilage Changes Induced by Treadmill Exercise in Rats.

This study aims to investigate the earliest alterations of bone and cartilage tissues as a result of different exercise protocols in the knee joint of Wistar rats. We hypothesize that pretraining to a continuous intense running protocol would protect the animals from cartilage degeneration. Three groups of animals were used: (i) an adaptive (pretraining) running group that ran for 8 weeks with gradually increasing velocity and time of running followed by a constant running program (6 weeks of 1.12 km/hour running per day); (ii) a non-adaptive running (constant running) group that initially rested for 8 weeks followed by 6 weeks of constant running; and (iii) a non-running (control) group. At weeks 8, 14, and 20 bone and cartilage were analyzed. Both running groups developed mild symptoms of cartilage irregularities, such as chondrocyte hypertrophy and cell clustering in different cartilage zones, in particular after the adaptive running protocol. As a result of physical training in the adaptive running exercise a dynamic response of bone was detected at week 8, where bone growth was enhanced. Conversely, the thickness of epiphyseal trabecular and subchondral bone (at week 14) was reduced due to the constant running in the period between 8 and 14 weeks. Finally, the intermediate differences between the two running groups disappeared after both groups had a resting period (from 14 to 20 weeks). The adaptive running group showed an increase in aggrecan gene expression and reduction of MMP2 expression after the initial 8 weeks running. Thus, the running exercise models in this study showed mild bone and cartilage/chondrocyte alterations that can be considered as early-stage osteoarthritis. The pretraining adaptive protocol before constant intense running did not protect from mild cartilage degeneration. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Multi-scale imaging techniques to investigate solute transport across articular cartilage.

As articular cartilage is an avascular tissue, the transport of nutrients and cytokines through the tissue is essential for the health of cells, i.e. chondrocytes. Transport of specific contrast agents through cartilage has been investigated to elucidate cartilage quality. In laboratory, pre-clinical and clinical studies, imaging techniques such as magnetic imaging resonance (MRI), computed tomography (CT) and fluorescent microscopy have been widely employed to visualize and quantify solute transport in cartilage. Many parameters related to the physico-chemical properties of the solute, such as molecular weight, net charge and chemical structure, have a profound effect on the transport characteristics. Information on the interplay of the solute parameters with the imaging-dependent parameters (e.g. resolution, scan and acquisition time) could assist in selecting the most optimal imaging systems and data analysis tools in a specific experimental set up. Here, we provide a comprehensive review of various imaging systems to investigate solute transport properties in articular cartilage, by discussing their potentials and limitations. The presented information can serve as a guideline for applications in cartilage imaging and therapeutics delivery and to improve understanding of the set-up of solute transport experiments in articular cartilage.

Phosphate Functional Groups Improve Oligo[(Polyethylene Glycol) Fumarate] Osteoconduction and BMP-2 Osteoinductive Efficacy.

Off-the-shelf availability in large quantities, drug delivery functionality, and modifiable chemistry and mechanical properties make synthetic polymers highly suitable candidates for bone grafting. However, most synthetic polymers lack the ability to support cell attachment, proliferation, migration, and differentiation, and ultimately tissue formation. Incorporating anionic peptides into the polymer that mimics acidic proteins, which contribute to biomineralization and cellular attachment, could enhance bone formation. Therefore, this study investigates the effect of a phosphate functional group on osteoconductivity and BMP-2-induced bone formation in an injectable and biodegradable oligo[(polyethylene glycol) fumarate] (OPF) hydrogel. Three types of OPF hydrogels were fabricated using 0%, 20%, or 40% Bis(2-(methacryloyloxy)ethyl) phosphate creating unmodified OPF-noBP and phosphate-modified OPF-BP20 and OPF-BP40, respectively. To account for the osteoinductive effect of various BMP-2 release profiles, two different release profiles (i.e., different ratios of burst and sustained release) were obtained by varying the BMP-2 loading method. To investigate the osteoconductive effect of phosphate modification, unloaded OPF composites were assessed for bone formation in a bone defect model after 3, 6, and 9 weeks. To determine the effect of the hydrogel phosphate modification on BMP-2-induced bone formation, BMP-2 loaded OPF composites with differential BMP-2 release were analyzed after 9 weeks of subcutaneous implantation in rats. The phosphate-modified OPF hydrogels (OPF-BP20 and OPF-BP40) generated significantly more bone in an orthotopic defect compared to the unmodified hydrogel (OPF-noBP). Furthermore, the phosphate functionalized surface-enhanced BMP-2-induced ectopic bone formation regardless of the BMP-2 release profile. In conclusion, this study clearly shows that phosphate functional groups improve the osteoconductive properties of OPF and enhanced BMP-2-induced bone formation. Therefore, functionalizing hydrogels with phosphate groups by crosslinking monomers into the hydrogel matrix could provide a valuable method for improving polymer characteristics and holds great promise for bone tissue engineering.

Metabolic dysregulation accelerates injury-induced joint degeneration, driven by local inflammation; an in vivo rat study.

Evidence is growing for the existence of an obesity-related phenotype of osteoarthritis in which low-grade inflammation and a disturbed metabolic profile play a role. The contribution of an obesity-induced metabolic dysbalance to the progression of the features of osteoarthritis upon mechanically induced cartilage damage was studied in a rat in vivo model. Forty Wistar rats were randomly allocated 1:1 to a standard diet or a high-fat diet. After 12 weeks, in 14 out of 20 rats in each group, cartilage was mechanically damaged in the right knee joint. The remaining six animals in each group served as controls. After a subsequent 12 weeks, serum was collected for metabolic state, subchondral bone changes assessed by µCT imaging, osteoarthritis severity determined by histology, and macrophage presence assessed by CD68 staining. The high-fat diet increased statistically all relevant metabolic parameters, resulting in a dysmetabolic state and subsequent synovial inflammation, whereas cartilage degeneration was hardly influenced. The high-fat condition in combination with mechanical cartilage damage resulted in a clear statistically significant progression of the osteoarthritic features, with increased synovitis and multiple large osteophytes. Both the synovium and osteophytes contained numerous CD68 positive cells. It is concluded that a metabolic dysbalance due to a high-fat diet increases joint inflammation without cartilage degeneration. The dysmetabolic state clearly accelerates progression of osteoarthritis upon surgically induced cartilage damage supported by inflammatory responses as demonstrated by histology and increased CD68 expressing cells localized on the synovial membrane and osteophytes. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:881-890, 2018.

Non-enzymatic cross-linking of collagen type II fibrils is tuned via osmolality switch.

An important aspect in cartilage ageing is accumulation of advanced glycation end products (AGEs) after exposure to sugars. Advanced glycation results in cross-links formation between the collagen fibrils in articular cartilage, hampering their flexibility and making cartilage more brittle. In the current study, we investigate whether collagen cross-linking after exposure to sugars depends on the stretching condition of the collagen fibrils. Healthy equine cartilage specimens were exposed to l-threose sugar and placed in hypo-, iso-, or hyper-osmolal conditions that expanded or shrank the tissue and changed the 3D conformation of collagen fibrils. We applied micro-indentation tests, contrast enhanced micro-computed tomography, biochemical measurement of pentosidine cross-links, and cartilage surface color analysis to assess the effects of advanced glycation cross-linking under these different conditions. Swelling of extracellular matrix due to hypo-osmolality made cartilage less susceptible to advanced glycation, namely, the increase in effective Young's modulus was approximately 80% lower in hypo-osmolality compared to hyper-osmolality and pentosidine content per collagen was 47% lower. These results indicate that healthy levels of glycosaminoglycans not only keep cartilage stiffness at appropriate levels by swelling and pre-stressed collagen fibrils, but also protect collagen fibrils from adverse effects of advanced glycation. These findings highlight the fact that collagen fibrils and therefore cartilage can be protected from further advanced glycation ("ageing") by maintaining the joint environment at sufficiently low osmolality. Understanding of mechanochemistry of collagen fibrils provided here might evoke potential ageing prohibiting strategies against cartilage deterioration. © 2018 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:1929-1936, 2018.

Anterior longitudinal ligament in diffuse idiopathic skeletal hyperostosis: Ossified or displaced?

Diffuse idiopathic skeletal hyperostosis (DISH) is often theorized to be an ossification of the anterior longitudinal ligament (ALL). Using computed tomography (CT) imaging and cryomacrotome sectioning, we investigated the spatial relationship between the ALL and newly formed bone in DISH to test this hypothesis. In the current study, four human cadaveric spines diagnosed with DISH using CT imaging were frozen and sectioned using a cryomacrotome. Photographs were obtained of the specimen at 125 µm intervals. Manual segmentations of the ALL on cryomacrotome photographs were projected onto the three-dimensional reconstructed CT scans. The presence and location of newly formed bone were assessed in relationship to the location of the ALL. The ALL could be identified and segmented on the photographs at all levels. The ALL was located at the midline at levels where no new bone had formed. At the locations where new bone had abundantly formed, the ALL was displaced towards to the contralateral side and not replaced by bony tissue. The displacement of the-morphologically normal appearing-ALL away from the newly formed bone implies that newly formed bone in DISH may not originate from the ALL. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 36:2491-2496, 2018.