The innovative design of orthopedic implants could play an important role in the development of life-lasting implants, by improving both primary and secondary implant fixations. The concept of meta-biomaterials aims to achieve a unique combination of mechanical, mass transport, and biological properties through optimized topological design of additively manufactured (AM) porous biomaterials. In this study, we primarily focused on a specific class of meta-biomaterials, namely auxetic meta-biomaterials. Their extraordinary behavior of lateral expansion in response to axial tension could potentially improve implant-bone contact in certain orthopedic applications. In this work, a multitude of auxetic meta-biomaterials were rationally designed and printed from Ti–6Al–4V using a commercially available laser powder bed fusion process called selective laser melting. The re-entrant hexagonal honeycomb unit cell was used as a starting point, which was then parametrically tuned to obtain a variety of mechanical and morphological properties. In this two-step study, the morphology and quasi-static properties of the developed meta-biomaterials were assessed using mechanical experiments accompanied with full-field strain measurements using digital image correlation. In addition, all our designs were computationally modelled using the finite element method. Our results showed the limits of the AM processes for the production of auxetic meta-biomaterials in terms of which values of the design parameters (e.g., re-entrant angle, relative density, and aspect ratio) could be successfully manufactured. We also found that the AM process itself imparts significant influence on the morphological and mechanical properties of the resulting auxetic meta-biomaterials. This further highlights the importance of experimental studies to determine the actual mechanical properties of such metamaterials. The elastic modulus and strength of many of our designs fell within the range of those reported for both trabecular and cortical bone. Unprecedented properties like these could be used to simultaneously address the different challenges faced in the mechanical design of orthopedic implants.

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.

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.

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 (Si₃N₄) 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 Si₃N₄. 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 Si₃N₄ group (52.6%) versus PEEK (27.9%; p = 0.2). There were no significant differences in BIC ratios between PEEK and Si₃N₄, and the biodynamic stability of the two groups was also comparable. The results suggest that Si₃N₄ spacers are not inferior to PEEK and they may be more effective in promoting arthrodesis.

Additively manufactured (AM) biodegradable metals with topologically ordered porous structures hold unprecedented promise as potential bone substitutes. The first reports on this type of biomaterials have just recently appeared in the literature. There is, however, no information available in the literature regarding their mechanical performance under cyclic loading or the interactions between biodegradation and cyclic loading. We therefore used selective laser melting (SLM) to fabricate porous magnesium alloy (WE43) scaffolds based on diamond unit cells. The microstructure of the resulting material was examined using electron back-scattered diffraction, scanning transmission electron microscopy, and X-ray diffraction. The fatigue behaviors of the material in air and in revised simulated body fluid (r-SBF) were evaluated and compared. Biodegradation decreased the fatigue strength of the porous material from 30% to 20% of its yield strength. Moreover, cyclic loading significantly increased its biodegradation rate. The mechanistic aspects of how biodegradation and cyclic loading interacted with each other on different scales were revealed as well. On the micro-scale, cracks initiated at biodegradation pits and propagated transgranularly. In addition, dislocations became more tangled after the fatigue tests. On the macro-scale, cracks preferred initiating at the strut junctions where tensile stress concentrations were present, as revealed by the finite element analysis of the porous material under compressive loading. Most of the cracks initiated in the struts were positioned on the periphery of the specimens. Furthermore, the biodegradation pattern was found to be location-dependent with more localized biodegradation occurring in the center of the specimens. Further improvements in the biodegradation-affected fatigue performance of the AM porous Mg alloy may therefore be realized by optimizing both the topological design of the porous structure and the laser-processing parameters that determine the microstructure of the SLM porous material.

Immune cells and their soluble factors regulate skeletal cells during normal bone regeneration and pathological bone formation. Bacterial infections can trigger immune responses that activate pro-osteogenic pathways, but these are usually overshadowed by osteolysis and concerns of systemic inflammation. The aim of this study was to determine whether the transient local inflammatory reaction to non-viable bacterial immune agonists could lead to favourable new bone formation. In a series of rabbit studies, as proof-of-concept, how tibial intramedullary injection of viable or killed bacterial species affected bone remodelling and new bone formation was determined. Application of killed bacteria led to considerable new bone formation after 4 weeks, without the prolonged systemic inflammation and exaggerated bone lysis seen with active infection. The osteo-immunomodulatory effects of various species of killed bacteria and the dose response relationship were subsequently screened in ectopically-implanted ceramic scaffolds. Histomorphometry after 8 weeks showed that a relatively low dose of killed bacteria enhanced ectopic bone induction. Moreover, lipoteichoic acid - the bacterial cell-wall derived toll-like-receptor (TLR)-2 activator - was identified as an osteo-stimulatory factor. Collectively, the data indicated that bacterial stimuli could be harnessed to stimulate osteogenesis, which occurs through a synergy with osteoinductive signals. This finding holds promise for the use of non-viable bacteria, bacterial antigens, or their simplified analogues as immuno-modulatory bone regenerating tools in bone biomaterials.

The main challenge in bone morphogenic protein 2 (BMP-2)-based application lies in finding strategies that prolong its effective period as it has a short biological half-life. Several BMP-2 release profiles have shown to enhance bone formation at various application sites. However, it remains to be determined which BMP-2 release profile best augments bone formation and whether this effect is location dependent. Therefore, the aim of this study was to investigate the effect of BMP-2 release from oligo[(polyethylene glycol) fumarate] bis(2-(methacryloyloxy)ethyl) phosphate (OPF-BP) composites on the osteoinductive efficacy at ectopic versus orthotopic application. By varying the BMP-2 loading method, three different OPF-BP composites were created with varied release profiles. The composites were compared with unloaded OPF-BP as negative control, and to the clinically used Infuse ^® absorbable collagen sponge (ACS) as positive control. Bone formation was assessed by microcomputed tomography after 9 weeks of subcutaneous implantation and 3, 6, and 9 weeks of orthotopic implantation in rats (n = 48). Whereas a BMP-2 burst release of >49% generated significantly more bone compared with sustained release (burst release <30%) at the subcutaneous implantation site, differential release did not affect bone formation at the orthotopic site. Furthermore, all BMP-2 containing OPF-BP composites showed significantly more bone formation compared with ACS in the orthotopic implantation site. In conclusion, this study clearly shows that the osteoinductive effect of different BMP-2 release profiles is location dependent. In addition, more bone formation in OPF-BP compared with ACS at both application sites emphasizes the role of biomaterials as a scaffold to achieve proper bone tissue formation. 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.

Since Galileo's days the effect of size on the anatomical characteristics of the structural elements of the body has been a subject of interest. However, the effects of scaling at tissue level have received little interest and virtually no data exist on the subject with respect to the osteochondral unit in the joint, despite this being one of the most lesion-prone and clinically relevant parts of the musculoskeletal system. Imaging techniques, including Fourier transform infrared imaging, polarized light microscopy and micro computed tomography, were combined to study the response to increasing body mass of the osteochondral unit. We analyzed the effect of scaling on structural characteristics of articular cartilage, subchondral plate and the supporting trabecular bone, across a wide range of mammals at microscopic level. We demonstrated that, while total cartilage thickness scales to body mass in a negative allometric fashion, thickness of different cartilage layers did not. Cartilage tissue layers were found to adapt to increasing loads principally in the deep zone with the superficial layers becoming relatively thinner. Subchondral plate thickness was found to have no correlation to body mass, nor did bone volume fraction. The underlying trabecular bone was found to have thicker trabeculae (r = 0.75, p < 0.001), as expected since this structure carries most loads and plays a role in force mitigation. The results of this study suggest that the osteochondral tissue structure has remained remarkably preserved across mammalian species during evolution, and that in particular, the trabecular bone carries the adaptation to the increasing body mass.

Additively manufactured (AM) functionally graded porous metallic biomaterials offer unique opportunities to satisfy the contradictory design requirements of an ideal bone substitute. However, no functionally graded porous structures have ever been 3D-printed from biodegradable metals, even though biodegradability is crucial both for full tissue regeneration and for the prevention of implant-associated infections in the long term. Here, we present the first ever report on AM functionally graded biodegradable porous metallic biomaterials. We made use of a diamond unit cell for the topological design of four different types of porous structures including two functionally graded structures and two reference uniform structures. Specimens were then fabricated from pure iron powder using selective laser melting (SLM), followed by experimental and computational analyses of their permeability, dynamic biodegradation behavior, mechanical properties, and cytocompatibility. It was found that the topological design with functional gradients controlled the fluid flow, mass transport properties and biodegradation behavior of the AM porous iron specimens, as up to 4-fold variations in permeability and up to 3-fold variations in biodegradation rate were observed for the different experimental groups. After 4 weeks of in vitro biodegradation, the AM porous scaffolds lost 5–16% of their weight. This falls into the desired range of biodegradation rates for bone substitution and confirms our hypothesis that topological design could indeed accelerate the biodegradation of otherwise slowly degrading metals, like iron. Even after 4 weeks of biodegradation, the mechanical properties of the specimens (i.e., E = 0.5–2.1 GPa, σ_y = 8–48 MPa) remained within the range of the values reported for trabecular bone. Design-dependent cell viability did not differ from gold standard controls for up to 48 h. This study clearly shows the great potential of AM functionally graded porous iron as a bone substituting material. Moreover, we demonstrate that complex topological design permits the control of mechanical properties, degradation behavior of AM porous metallic biomaterials. Statement of Significance: No functionally graded porous structures have ever been 3D-printed from biodegradable metals, even though biodegradability is crucial both for full tissue regeneration and for the prevention of implant-associated infections in the long term. Here, we present the first report on 3D-printed functionally graded biodegradable porous metallic biomaterials. Our results suggest that topological design in general, and functional gradients in particular can be used as an important tool for adjusting the biodegradation behavior of AM porous metallic biomaterials. The biodegradation rate and mass transport properties of AM porous iron can be increased while maintaining the bone-mimicking mechanical properties of these biomaterials. The observations reported here underline the importance of proper topological design in the development of AM porous biodegradable metals.

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.

Additive manufacturing (AM) techniques enable fabrication of bone-mimicking meta-biomaterials with unprecedented combinations of topological, mechanical, and mass transport properties. The mechanical performance of AM meta-biomaterials is a direct function of their topological design. It is, however, not clear to what extent the material type is important in determining the fatigue behavior of such biomaterials. We therefore aimed to determine the isolated and modulated effects of topological design and material type on the fatigue response of metallic meta-biomaterials fabricated with selective laser melting. Towards that end, we designed and additively manufactured Co-Cr meta-biomaterials with three types of repeating unit cells and three to four porosities per type of repeating unit cell. The AM meta-biomaterials were then mechanically tested to obtain their normalized S-N curves. The obtained S-N curves of Co-Cr meta-biomaterials were compared to those of meta-biomaterials with same topological designs but made from other materials, i.e. Ti-6Al-4V, tantalum, and pure titanium, available from our previous studies. We found the material type to be far more important than the topological design in determining the normalized fatigue strength of our AM metallic meta-biomaterials. This is the opposite of what we have found for the quasi-static mechanical properties of the same meta-biomaterials. The effects of material type, manufacturing imperfections, and topological design were different in the high and low cycle fatigue regions. That is likely because the cyclic response of meta-biomaterials depends not only on the static and fatigue strengths of the bulk material but also on other factors that may include strut roughness, distribution of the micro-pores created inside the struts during the AM process, and plasticity. Statement of significance: Meta-biomaterials are a special class of metamaterials with unusual or unprecedented combinations of mechanical, physical (e.g. mass transport), and biological properties. Topologically complex and additively manufactured meta-biomaterials have been shown to improve bone regeneration and osseointegration. The mechanical properties of such biomaterials are directly related to their topological design and material type. However, previous studies of such biomaterials have largely neglected the effects of material type, instead focusing on topological design. We show here that neglecting the effects of material type is unjustified. We studied the isolated and combined effects of topological design and material type on the normalized S-N curves of metallic bone-mimicking biomaterials and found them to be more strongly dependent on the material type than topological design.

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.

Implant-associated infections (IAI) are often recurrent, expensive to treat, and associated with high rates of morbidity, if not mortality. We biofunctionalized the surface of additively manufactured volume-porous titanium implants using electrophoretic deposition (EPD) as a way to eliminate the peri-operative bacterial load and prevent IAI. Chitosan-based (Ch) coatings were incorporated with different concentrations of silver (Ag) nanoparticles or vancomycin. A full-scale in vitro and in vivo study was then performed to evaluate the antibacterial, immunogenic, and osteogenic activity of the developed implants. In vitro, Ch + vancomycin or Ch + Ag coatings completely eliminated, or reduced the number of planktonic and adherent Staphylococcus aureus by up to 4 orders of magnitude, respectively. In an in vivo tibia intramedullary implant model, Ch + Ag coatings caused no adverse immune or bone response under aseptic conditions. Following Staphylococcus aureus inoculation, Ch + vancomycin coatings reduced the implant infection rate as compared to chitosan-only coatings. Ch + Ag implants did not demonstrate antibacterial effects in vivo and even aggravated infection-mediated bone remodeling including increased osteoclast formation and inflammation-induced new bone formation. As an explanation for the poor antibacterial activity of Ch + Ag implants, it was found that antibacterial Ag concentrations were cytotoxic for neutrophils, and that non-toxic Ag concentrations diminished their phagocytic activity. This study shows the potential of EPD coating to biofunctionalize porous titanium implants with different antibacterial agents. Using this method, Ag-based coatings seem inferior to antibiotic coatings, as their adverse effects on the normal immune response could cancel the direct antibacterial effects of Ag nanoparticles. Statement of Significance: Implant-associated infections (IAI) are a clinical, societal, and economical burden. Surface biofunctionalization approaches can render complex metal implants with strong local antibacterial action. The antibacterial effects of inorganic materials such as silver nanoparticles (Ag NPs) are often highlighted under very confined conditions in vitro. As a novelty, this study also reports the antibacterial, immunogenic, and osteogenic activity of Ag NP-coated additively-manufactured titanium in vivo. Importantly, it was found that the developed coatings could impair the normal function of neutrophils, the most important phagocytic cells protecting us from IAI. Not surprisingly, the Ag NP-based coatings were outperformed by an antibiotic-based coating. This emphasizes the importance of also targeting implant immune-modulatory functions in future coating strategies against IAI.

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.

This experimental work focused on the sensor selection for the development of a needle-like instrument to treat small isolated cartilage defects with hydrogels. The aim was to identify the most accurate and sensitive imaging method to determine the location and size of defects compared to a gold standard (µCT). Only intravascular ultrasound imaging (IVUS) vs. optical coherent tomography (OCT) were looked at, as they fulfilled the criteria for integration in the needle design. An in-vitro study was conducted on six human cadaveric tali that were dissected and submerged in saline. To simulate the natural appearance of cartilage defects, three types of defects were created via a standardised protocol: osteochondral defects (OCD), chondral defects (CD) and cartilage surface fibrillation (CSF), all sized between 0.1 and 3 mm in diameter. The detection rate by two observers for all diameters of OCD were 80, 92 and 100% with IVUS, OCT and µCT, for CD these were 60, 83 and 97%, and for CSF 0, 29 and 24%. Both IVUS and OCT can detect the presence of OCD and CD accurately if they are larger than 2 mm in diameter, and OCT can detect fibrillated cartilage defects larger than 3 mm in diameter. A significant difference between OCT–µCT and IVUS–µCT was found for the diameter error (p = 0.004) and insertion depth error (p = 0.002), indicating that OCT gives values closer to reference µCT. The OCT imaging technique is more sensitive to various types and sizes of defects and has a smaller diameter, and is therefore preferred for the intended application.