Autologous bone grafts are commonly used to repair defects in skeletal tissue, however, due to their limited supply there is a clinical need for alternatives. Synthetic ceramics present a promising option but currently lack biological activity to stimulate bone regeneration. One potential approach to address this limitation is the incorporation of immunomodulatory agents. In this study, we investigate the application of microbial stimuli to stimulate bone formation. Three different microbial stimuli were incorporated in a biphasic calcium phosphate (BCP) ceramic: Bacille Calmette-Guérin (BCG), gamma-irradiated Staphylococcus aureus (γi-S. aureus), or γi-Candida albicans (γi-C. Albicans). The constructs were then implanted in both a rabbit posterolateral spinal fusion (PLF) and an intramuscular implant model for 10 weeks and compared to a nonstimulated control construct. For the PLF model, the formation of a bony bridge was evaluated by manual palpation, micro computed tomography, and histology. While complete fusion was not observed, the BCG condition was most promising with higher manual stiffness and almost twice as much bone volume in the central fusion mass compared to the control (9 ± 4.4% bone area vs. 4.6 ± 2.3%, respectively). Conversely, the γi-S. aureus or γi-C. albicans appeared to inhibit bone formation (1.4 ± 1.4% and 1.2 ± 0.6% bone area). Bone induction was not observed in any of the intramuscular implants. This study indicates that incorporating immunomodulatory agents in ceramic bone substitutes can affect bone formation, which can be positive when selected carefully. The readily available and clinically approved BCG showed promising results, which warrants further research for clinical translation.

@en

Background: Lumbar interbody fusion (IF) is a common procedure to fuse the anterior spine. However, a lack of consensus on image-based fusion assessment limits the validity and comparison of IF studies. This systematic review aims to (1) report on IF assessment strategies and definitions and (2) summarize available literature on the diagnostic reliability and accuracy of these assessments. Methods: Two searches were performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Search 1 identified studies on adult lumbar IF that provided a detailed description of image-based fusion assessment. Search 2 analyzed studies on the reliability of specific fusion criteria/classifications and the accuracy assessed with surgical exploration. Results: A total of 442 studies were included for search 1 and 8 studies for search 2. Fusion assessment throughout the literature was highly variable. Eighteen definitions and more than 250 unique fusion assessment methods were identified. The criteria that showed most consistent use were continuity of bony bridging, radiolucency around the cage, and angular motion <5°. However, reliability and accuracy studies were scarce. Conclusion: This review highlights the challenges in reaching consensus on IF assessment. The variability in IF assessment is very high, which limits the translatability of studies. Accuracy studies are needed to guide innovations of assessment. Future IF assessment strategies should focus on the standardization of computed tomography-based continuity of bony bridging. Knowledge from preclinical and imaging studies can add valuable information to this ongoing discussion.

@en

Macrophages are key regulators in bone repair and regeneration. Recent studies have shown that long-term epigenetic changes and metabolic shifts occur during specific immune training of macrophages that affect their functional state, resulting in heightened (trained) or reduced (tolerant) responses upon exposure to a second stimulus. This is known as innate immune memory. Here, we study the impact of macrophages’ memory trait on osteoblast differentiation of human mesenchymal stromal cells (hMSCs) and osteoclast differentiation. An in vitro trained immunity protocol of monocyte-derived macrophages was employed using inactivated Candida albicans and Bacillus Calmette–Guérin (BCG) to induce a ‘trained’ state and Pam3CSK4 (PAM) and Lipopolysaccharides (LPS) to induce a ‘tolerance’ state. Macrophages were subsequently cocultured with hMSCs undergoing osteogenic differentiation during either resting (unstimulated) or inflammatory conditions (restimulated with LPS). Alkaline phosphatase activity, mineralization, and cytokine levels (TNF, IL-6, oncostatin M and SDF-1α) were measured. In addition, macrophages underwent osteoclast differentiation. Our findings show that trained and tolerized macrophages induced opposing results. Under resting conditions, BCG-trained macrophages enhanced ALP levels (threefold), while under inflammatory conditions this was found in the LPS-tolerized macrophages (fourfold). Coculture of hMSCs with trained macrophages showed mineralization while tolerized macrophages inhibited the process under both resting and inflammatory conditions. While osteoclast differentiation was not affected in trained-macrophages, this ability was significantly loss in tolerized ones. This study further confirms the intricate cross talk between immune cells and bone cells, highlighting the need to consider this interaction in the development of personalized approaches for bone regenerative medicine. Graphical Abstract: (Figure presented.).

@en

For decades, the advantages of rapid prototyping for clinical use have been recognized. However, demonstrations of potential solutions to treat spinal problems that cannot be solved otherwise are scarce. In this paper, we describe the development, regulatory process, and clinical application of two types of patient specific 3D-printed devices that were developed at an in-house 3D point-of-care facility. This 3D lab made it possible to elegantly treat patients with spinal problems that could not have been treated in a conventional manner. The first device, applied in three patients, is a printed nylon drill guide, with such accuracy that it can be used for insertion of cervical pedicle screws in very young children, which has been applied even in semi-acute settings. The other is a 3D-printed titanium spinal column prosthesis that was used to treat progressive and severe deformities due to lysis of the anterior column in three patients. The unique opportunity to control size, shape, and material characteristics allowed a relatively easy solution for these patients, who were developing paraplegia. In this paper, we discuss the pathway toward the design and final application, including technical file creation for dossier building and challenges within a point-of-care lab.

@en

Therapeutic pathogen recognition receptor (PRR) ligands are reaching clinical practice following their ability to skew the immune response in a specific direction. We investigated the effects of various therapeutic PRR ligands on bone cell differentiation and inflammation. Following stimu-lation, alkaline phosphatase (ALP) activity (Day 10), osteocalcin, osteonectin expression (Day 14), and calcium deposition (Day 21) were quantified in bone marrow-derived human mesenchymal stem cells (hMSCs). The osteoclastogenic response was determined by measuring tartrate-resistant acid phosphate (TRAP) activity in human monocytes. TNF-α, IL-6, IL-8, and IL-10 expressions were measured by enzyme-linked immunosorbent assay as an indicator of the ligands’ inflammatory properties. We found that nucleic acid-based ligands Poly(I:C) and CpG ODN C increased early ALP activity in hMSCs by 4-fold without affecting osteoclast formation. These ligands did not enhance expression of the other, late osteogenic markers. MPLA, Curdlan, and Pam3CSK4 did not affect osteogenic differentiation, but inhibited TRAP activity in monocytes, which was associated with increased expression of all measured cytokines. Nucleic acid-based ligands are identified as the most promising osteo-immunomodulators, as they favor early osteogenic differentiation without inducing an exaggerated immune-cell mediated response or interfering in osteoclastogenesis and thus can be potentially harnessed for multifunctional coatings for bone biomaterials.

@en

Background: Additive manufacturing or three-dimensional (3D) printing of metal implants can provide novel solutions for difficult-to-treat conditions, yet legislation concerning patient-specific implants complicates the implementation of these techniques in daily practice. In this Article, we share our acquired knowledge of the logistical and legal challenges associated with the use of patient-specific 3D-printed implants to treat spinal instabilities. Methods: Two patients with semiurgent cases of spinal instability presented to our hospital in the Netherlands. In case 1, severe kyphotic deformity of the thoracic spine due to neurofibromatosis type 1 had led to incomplete paralysis, and a strong metallic strut extending from C6 to T11 was deemed necessary to provide long-term anterior support. In case 2, the patient presented with progressive paralysis caused by cervicothoracic dissociation due to vanishing bone disease. As the C5–T1 vertebral bodies had mostly vanished, an implant spanning the anterior spine from C4 to T2 was required. Because of the complex and challenging nature of both cases, conventional approaches were deemed inadequate; instead, patient-specific implants were designed with use of CT scans and computer-aided design software, and 3D printed in titanium with direct metal printing. For each implant, to ensure patient safety, a comprehensive technical file (describing the clinical substantiation, technical and design considerations, risk analysis, manufacturing process, and labelling) was produced in collaboration with a university department certified for the development and manufacturing of medical devices. Because the implants were categorised as custom-made or personalised devices under the EU Medical Device Regulation, the usual procedures for review and approval of medical devices by a notified body were not required. Finite-element analyses, compression strength tests, and cadaveric experiments were also done to ensure the devices were safe to use. Findings: The planning, design, production, and insertion of the 3D-printed personalised implant took around 6 months in the first patient, but, given the experience from the first case, only took around 6 weeks in the second patient. In both patients, the surgeries went as planned and good positioning of each implant was confirmed. Both patients were discharged home within 1 week after the surgery. In the first patient, a fatigue fracture occured in one of the conventional posterior fusion rods after 10 months, which we repaired, without any deformation of the spine or signs of failure of the personalised implant observed. No other adverse events occurred up to 25 months of follow-up in case 1 and 6 months of follow-up in case 2. Interpretation: Patient-specific treatment approaches incorporating 3D-printed implants can be helpful in carefully selected cases when conventional methods are not an option. Comprehensive and efficient interactions between medical engineers and physicians are essential to establish well designed frameworks to navigate the logistical and regulatory aspects of technology development to ensure the safety and legal validity of patient-specific treatments. The framework described here could encourage physicians to treat (once untreatable) patients with novel personalised techniques. Funding: Interreg VA Flanders—The Netherlands programme, Applied and Engineering Sciences research programme, the Netherlands Organisation for Scientific Research, and the Dutch Arthritis Foundation Video Abstract.

@en

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.

@en

Summary: Extracorporeal shockwave therapy showed a pronounced effect on bone mass in previous animal studies. We showed in this pilot study that a single treatment with unfocused shockwave therapy in unselected patients does not show side effects. Although our study did not show any effect of shockwave on BMD, the limited sample size does not definitively exclude this and a study with 174 subjects per group would be needed to show an effect size of 0.3 with a power of 80%. Purpose: Unfocused extracorporeal shockwave therapy might stimulate bone formation to reduce the fracture risk. In this study, we assessed the safety of unfocused extracorporeal shockwave therapy and its effects on bone mass. Methods: A clinical pilot study with twelve female patients free of bone disease undergoing elective surgery of the lower extremity or elective spinal surgery under general anesthesia received 3.000 electrohydraulic-generated unfocused extracorporeal shockwaves (energy flux density 0.3 mJ/mm²) to one distal forearm. The contralateral forearm served as a control. We examined the effect on bone mass with the use of repeated dual energy X-ray absorptiometry measurements and we measured patient discomfort around the therapy. Results: No difference in bone mineral content and density was measured 6 and 12 weeks after therapy. shockwave therapy occasionally caused transient erythema or mild hematoma, but no discomfort in daily life or (late) adverse events. Conclusions: Unfocused extracorporeal shockwave therapy is a safe treatment, but no increase in bone mass on the forearm was found at 0.3 mJ/mm² energy flux density. In this study, we were not able to demonstrate that a single treatment with unfocused shockwave therapy in unselected patients had any effect in terms of bone mineral density (BMD) or bone mineral content (BMC). A power analysis indicated that 174 patients per group are required to show an effect size of 0.3 with a power of 80%.

@en

5 jaar geleden beschreven wij de opkomst van 3D-printen in de geneeskunde. Het betrof het 3D-printen van anatomische structuren, patiëntspecifieke boorgeleiders, zaagmallen en implantaten en het printen van levende cellen, groeifactoren en biomaterialen (‘bioprinten’). Chirurgen maken steeds vaker gebruik van de mogelijkheden van 3D-printen ter voorbereiding op operaties bij patiënten met een ingewikkelde anatomie. Aan de hand van tastbare 3D-modellen kunnen chirurgen de operatie beter voorbereiden en bespreken met de patiënt. Ook kunnen zij de 3D-modellen gebruiken als hulpmiddel bij de training van jonge chirurgen. Steeds vaker worden permanente titanium implantaten geprint. Bioprinten staat nog in de kinderschoenen en kent nog geen directe klinische toepassingen. Zoals wij 5 jaar geleden al voorspelden, moeten er voor de brede klinische toepassing van gebioprinte producten nog vele hordes genomen worden.@en

Screw fixation in osteoporotic patients is becoming an increasing problem in orthopaedic surgery as deterioration of cortical and cancellous bone hamper biomechanical stability and screw fixation. This might result in delayed weight-bearing or failure of instrumentation. We hypothesized that local peri-operative shock wave treatment can optimize osseointegration and subsequent screw fixation. In eight female Wistar rats, two cancellous and two cortical bone screws were implanted in both femora and tibiae. Immediately after implantation, 3.000 unfocused extracorporeal shock waves (energy flux density 0.3 mJ/mm2) were applied to one side. The other side served as non-treated internal control. Evaluation of osseointegration was performed after 4 weeks with the use of microCT scanning, histology with fluorochrome labeling, and pull-out tests of the screws. Four weeks after extracorporeal shock wave treatment, treated legs exhibited increased bone formation and screw fixation around cortical screws as compared to the control legs. This was corroborated by an increased pull-out of the shock wave treated cortical screws. The cancellous bone screws appeared not to be sensitive for shock wave treatment. Formation of neocortices after shock wave therapy was observed in three of eight animals. Furthermore, de novo bone formation in the bone marrow was observed in some animals. The current study showed bone formation and improved screw fixation as a result of shock wave therapy. New bone was also formed at locations remote from the screws, hence, not contributing to screw fixation. Further, research is warranted to make shock wave therapy tailor-made for fracture fixation@en

Pathologic conditions associated with bone formation can serve as models to identify bone-promoting mediators. The inflammatory response to bacterial infections generally leads to osteolysis and impaired bone healing, but
paradoxically, it can also have pro-osteogenic effects. As a potential model to investigate pro-osteogenic stimuli, this study characterizes the bone formation in an established rabbit tibia model of periprosthetic infection. Our hypothesis
was that the infection with Staphylococcus aureus (S. aureus) correlates with bone formation as a response to local inflammation. Fluorochromes showed excessive subperiosteal bone formation in infected tibiae, starting the first
week and continuing throughout the study period. Despite the observed cortical lysis on micro-CT after 28 days, infection resulted in a twofold higher bone volume in the proximal tibiae compared to uninfected controls. The
ipsilateral fibulae, nor the contralateral fibulae or tibiae were affected by infection. Next, we sought to confine the cause of stimulated bone formation to the isolated S. aureus cell wall. In absence of virulent bacterial infection, the S.
aureus cell wall extract induced bone in a more favorable way without cortical lysis. This suggests that the sterile inflammatory reaction to bacterial antigens may be harnessed for bone regenerative purposes. Future investigations
in this rabbit tibia model can lead to further identification of effective stimuli for clinical application.@en