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Back pain, affecting 80% of the population, significantly strains the healthcare system. In European countries, spine-related hospital discharges account for 14.2% to 45.6% of all musculoskeletal disease discharges. Conservative treatments like medication and physical therapy are generally preferred, but surgical intervention may be necessary for some. Spinal surgeries often involve implants, such as spinal cages, spinal instrumentation, or total disc replacements, used to treat abnormal spinal curvatures or intervertebral disc degeneration.

Despite their widespread use, spinal implants face challenges such as failed vertebral fusion, infections, and implant failure, which can release harmful ions and particles. Researchers are developing new materials with antibacterial properties and improved interaction with bone tissue. Innovations include wear-resistant coatings to prevent metal ion release and biodegradable materials that the body gradually replaces, reducing infection risks and the need for revision surgeries. However, these advances present challenges. Degradation by-products can migrate more easily to other parts of the body and may elicit unwanted biological responses.

The primary aim of this thesis was to investigate the biological effects of these degradation products from an in vitro perspective. This involved using several relevant cell types and examining morphological and functional changes. A composite of calcium phosphate and polylactic acid was initially examined for spinal fusion. The cell response to the degradation products was comparable to those of a clinically successful calcium phosphate, showing no negative impact on preosteoblast cells. Additionally, silicon nitride (SiN) coatings, known for their wear resistance properties, were explored. The incorporation of additional elements into SiN coatings was studied to enhance stability and durability. It was found that fibroblast and microglial cells tolerated the ions and particles released during degradation similarly to current orthopedic materials. Lastly, the effects of particles from spinal implants on glial cells were evaluated. While most particles did not trigger inflammation, high doses of SiN particles negatively affected microglial cells, reducing their ability to neutralize infectious agents. This highlights the need for further research to fully understand the biological safety of silicon nitride in spinal implants.

In summary, this thesis expands the understanding of the biological responses to spinal implant degradation products, aiding the development of safer and more effective implants.

Composite cranial implants composed of calcium phosphate cement (CPC) and titanium have improved cranioplasty procedures outcomes. The patient’s bone grows back and replaces the CPC, while the titanium remains. Therefore, fully resorbable implants could allow for an increased use in paediatric surgery and a reduced need for revision surgeries. We propose replacing the titanium by a resorbable, biocompatible, 3D-printable polymer with suitable mechanical properties and degradation rate. While the biological response to the individual materials has been thoroughly investigated in the literature, specific combinations of materials have not. These could give a different response due to e.g. pH variations. In this in vitro study, we investigate the degradation behaviour of CPC-poly-L-lactic acid (PLLA) structures and the effect of the degradation’s by-products on preosteoblastic cells.

Samples were prepared by moulding CPC around a 3D-printed dense PLLA beam, mimicking the current implants composed of a structural supporting mesh embedded in CPC tiles. The monetite CPC was obtained with β-tricalcium phosphate (βTCP) and monocalcium phosphate monohydrate (MCPM). Degradation tests were conducted in a physiologically relevant environment for parallel evaluation of cellular responses. Various techniques (e.g. Scanning Electron Microscopy, Differential Scanning Calorimetry, X-Ray Diffraction, Inductively Coupled Plasma Optical Emission Spectroscopy, pH monitoring) were applied for morphological, chemical, and thermal characterization of samples and extracts. MC3T3 preosteoblastic cells were cultured with inserts containing the samples, to gain insights into the critical early stages of bone healing and on the composite’s capability for osteoinduction. Over time, we measured cell viability (metabolic activity), progression towards osteoblastic differentiation (ALP expression and enzymatic activity), and matrix mineralization (to determine the differentiation efficacy of the osteoblasts).

This experiment was conducted to study candidate materials for new resorbable cranial implants, assessing their degradation processes and potential interactions, as well as the impact of degradation by-products on cell viability and osteogenic potential.

Injectable poly (methyl methacrylate) (PMMA) bone cements are widely used in orthopaedics to stabilize fractures and for implant fixation. However, bacterial attachment to bone cements leads to significant complications that can create a need for implant revision. Common attempts at reducing bacterial attachment are through the addition of antibiotics or antibacterial nanometals to the bone cements. However, clinical data is inconclusive on the effectiveness of antibiotic-loaded bone cements and a negative osteoblastic response has been reported for certain additive concentrations. There is therefore a need for an additive that can positively affect osteoblastic behaviour while inhibiting bacterial attachment. Silicon nitride (Si3N4) could be such an additive, with initial studies showing promise in achieving antipathogenic properties. The aim of this study was hence to investigate the possibility of creating a bone cement that can support osteoblast growth while reducing bacterial attachment by introducing silicon nitride powders into an injectable PMMA cement. To this end, commercially available bone cements were doped with 5%, 10% and 20% weight/weight (w/w) of Si3N4. Their mechanical properties were examined through compression testing and their radiopacity was evaluated through fluoroscopy imaging. The samples that fulfilled compressive strength requirements had their biological properties tested using Staphylococcus epidermidis bacteria for antibacterial properties and MC3T3-E1 preosteoblasts for the examination of cytotoxicity. Bone cements that were doped with up to 20% w/w Si3N4 were radiopaque (only 13% reduction in optical density compared to radiopaque controls) and retained their compressive strength (85.35 ± 2.1 MPa compared to 83.4 ± 1.9 MPa for the commercial cements), while significantly reducing bacterial attachment by more than 90% compared to commercial cements and achieving a similar level of preosteoblast metabolic activity. This study supports further evaluation of Si3N4 as an additive to injectable bone cements as a way to create mechanically stable, radiopaque, bacteriostatic bone cements that could improve osteointegration.

In cranioplasty procedures, composite cranial implants using calcium phosphate cement (CP) and 3D-printed titanium alloy have shown improved surgical outcomes [1,2]. While CP is replaced by the patient’s bone over time, the titanium remains.  A fully resorbable implant would enable expansion to applications in pediatric surgery and reduce the need for revision surgeries. We propose using a resorbable, biocompatible, and 3D-printable polymer with suitable mechanical properties and degradation rates instead of titanium. While biological responses to individual materials are well-studied, interactions within specific material combinations are less explored. This study investigates the degradation behavior of CP combined with poly-L-lactic acid (PLLA) [3,4] and its effects on preosteoblastic cells.

Implant-like samples were prepared by molding CP around a 3D-printed dense PLLA beam and tested in a physiologically relevant environment, enabling a concurrent assessment of cellular responses. Techniques including Scanning Electron Microscopy, X-Ray Diffraction, Inductively Coupled Plasma Optical Emission Spectroscopy and mass monitoring were used for characterization. MC3T3 preosteoblastic cells cultured with these samples provided insights into early bone healing and the composite's osteoinductive potential, with evaluations of cell viability, osteoblastic differentiation, and matrix mineralization. 

While limited degradation occurred over 28 days, changes in calcium and phosphorus levels influenced early biological responses, delaying cell proliferation, metabolic activity, and differentiation. However, by the end of the study, degradation by-products did not adversely affect cell metabolic activity or osteogenic potential. These findings suggest that the CP-PLLA composite structure holds promise and is comparable to the clinically established CP in terms of in vitro preosteoblastic cell response.

References

[1] L. Kihlström Burenstam Linder et al., World Neurosurgery, 2019, 122:e399–407. doi: 10.1016/j.wneu.2018.10.061.

[2] S. Lewin et al., Acta Biomaterialia, 2021, 128:502–13. doi: 10.1016/j.actbio.2021.04.015.

[3] E. Capuana et al., Polymers, 2022, 14(6):1153. doi: 10.3390/polym14061153.

[4] T. Ahlfeld et al., Biomaterials Science, 2023. doi: 10.1039/d2bm02071h.

Silicon nitride (SiN) coatings may reduce unwanted release of metal ions from metallic implants. However, as SiN slowly dissolves in aqueous solutions, additives that reduce this dissolution rate would likely increase the lifetime and functionality of implants. Adding iron (Fe) and carbon (C) permits tuning of the SiN coatings’ mechanical properties, but their effect on SiN dissolution rates, and their capacity to reduce metal ion release from metallic implant substrates, have yet to be investigated. Such coatings have recently been proposed for use in spinal implants; therefore, it is relevant to assess their impact on the viability of cells expected at the implant site, such as microglia, the resident macrophages of the central nervous system (CNS). To study the effects of Fe and C on the dissolution rate of SiN coatings, compositional gradients of Si, Fe and C in combination with N were generated by physical vapor deposition onto CoCrMo discs. Differences in composition did not affect the surface roughness or the release of Si, Fe or Co ions (the latter from the CoCrMo substrate). Adding Fe and C reduced ion release compared to a SiN reference coating, which was attributed to altered reactivity due to an increase in the fraction of stabilizing Si–C or Fe–C bonds. Extracts from the SiN coatings containing Fe and C were compatible with microglial viability in 2D cultures and 3D collagen hydrogels, to a similar degree as CoCrMo and SiN coated CoCrMo reference extracts. As Fe and C reduced the dissolution rate of SiN-coatings and did not compromise microglial viability, the capacity of these additives to extend the lifetime and functionality of SiN-coated metallic implants warrants further investigation.

Spinal implants have been used for decades to treat different spinal conditions. However, certain implant-related complications have been attributed to the release of particles and ions due to corrosion and wear triggering local immune responses including the release of pro-inflammatory cytokines, leading to local inflammation. The impact of these particles and ions on cells from the central nervous system (CNS) remains largely unknown, with few studies examining the effects on glial cells1. Indeed, the particles may migrate to adjacent nervous tissues and increasing our knowledge of the glial cell response is essential since they play a crucial role in maintaining tissue homeostasis and protecting the CNS. Most prior studies have used traditional 2D culture models; however, these lack the 3D spatial arrangement of cells found in tissues where they form important interactions with the extracellular matrix. The aim of this study was to employ an open source bioprinter2 to extrude hydrogels containing glial cells into which experimental implant debris can be introduced, enabling monitoring of cell viability and inflammatory responses by fluorescence microscopy. We have previously established that mono-cultures of microglia and astrocytes can be 3D cultured in collagen hydrogels, and their viability monitored using the caspase-3/7 apoptosis reporter and propidium iodide labelling for cell death. Applying a bioprinting strategy to produce these glial-laden constructs increases the reproducibility of these models, and allows the study of a wide range of types and concentrations of particles, resulting in a valuable tool to increase the knowledge about the biological response generated by particles from spinal implants.

INTRODUCTION: Wear and corrosion may lead to a release of particles and ions from spinal implants, which is a concern because of their potentially detrimental effect on the life span of the implant [1]. Silicon nitride-based coatings have been suggested as an option to reduce the release of metal ions from an implant. In addition, any particles produced will slowly dissolve, releasing only biocompatible ions [2]. It is of high interest to reduce the dissolution rate of the coating to ensure an adequate lifetime [3]. The present study aimed to assess the effect of Fe and C additions to silicon nitride coatings in terms of dissolution rate as well as the impact of the released ions on the in vitro neural cell response.

METHODS: Using a combinatorial approach, SiFeCN coatings were deposited on CoCr disc substrates by reactive sputtering in an in-house built equipment. The coatings were characterized in 9 points using x-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). The dissolution behaviour was evaluated by exposing the coated samples to cell media for 14 days. The obtained extracts were used to measure ion release with inductively coupled plasma - optical emission spectrometry (ICP-OES) and to assess cell viability of microglia (C8-B4 cell line) using the MTT assay. 

RESULTS: The XPS results showed compositional gradients of Si ranging from 35.0 to 47.3 at.%, Fe from 1.4 to 9.3 at.% and C from 4.5 to 13.9 at.%. SEM of focused ion beam (FIB) cross-sections revealed coating thicknesses between 427-534 nm. SEM of the coating after exposure showed substantial signs of dissolution with visibly increased porosity for the SiN coating, while the SiFeCN coatings appeared less affected. SiFeCN coatings appeared more affected by dissolution for increasing Si contents. The estimated dissolution rate of the SiN coating was 8.3 nm/day, while the rate of SiFeCN coatings was 5.2-6.8 nm/day. The ICP results showed a reduction in Co ions from the substrate in the coated samples compared to uncoated CoCr. Moreover, the levels of detected Si ions were lower for the SiFeCN compared to SiN reference. Indirect biocompatibility tests suggested that microglia cell viability was comparable for the SiFeCN coatings, the uncoated CoCr and the SiN coating.

DISCUSSION & CONCLUSIONS: The compositional gradients influenced the thickness of the coating, giving a slight thickness increase in the coatings with the increment of Si content. In addition, the ICP results showed the capability of the coating to act as a barrier to the release of ions from the substrate. Furthermore, the presence of Fe and C in the coating causes a decrease in the ion release from the coating, indicative of a lower dissolution rate, which was supported by the thickness measurements. The findings from this study indicate that using Fe and C as alloying elements can lower the dissolution rate of the silicon nitride-based coating while showing positive indications of biocompatibility on neural cells. Therefore, SiFeCN coatings merit further investigation as a future option for spinal implants.

REFERENCES: 1Y. Shimamura et al (2008) Spine. 33:351–355. 2M.  Pettersson et al (2016) ACS Biomater. Sci. Eng. 2:998–1004. 3C. Skjöldebrand et al (2022) Biomater Sci, 10:3757–3769.

ACKNOWLEDGEMENTS: This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 812765 and from the European Union’s Seventh Framework Programme (FP7/2007-2013), grant agreement GA-310477(LifeLongJoints).

Spinal implants have been used for several decades to relieve pain and stabilise the spine, however an increase in the life span of implants is required due to younger and more active patients requiring spinal surgery. Silicon nitride-based coatings have been suggested as an alternative to metallic implants to reduce the release of detrimental ion and particles. However, due to the coating’s dissolution in the presence of water, reducing the dissolution rate by altering the coating composition is of high interest to ensure an adequate lifetime. The aim of this study was to investigate the dissolution rate of silicon nitride coatings containing Fe and C and the effect of the ions released on in vitro neural cell response. 

SiFeCN coatings were deposited by reactive sputtering using a combinatorial approach for efficient testing of different compositions. Compositional gradients were obtained for the investigated elements. SEM of the coated samples after exposure to cell media displayed stronger signs of dissolution on the SiN reference than the alloyed coatings. The addition of Fe and C decreased the ion release of the coating itself compared to the SiN coating. Indirect biocompatibility tests suggested that microglial cell viability was comparable to that of CoCrMo reference samples and SiN coatings. 

In conclusion, the results indicate the possibility of decreasing the dissolution rate of SiN coatings by the addition of Fe and C, while maintaining the biocompatibility as confirmed by the cytotoxicity tests on neural cells. Therefore, SiFeCN coatings merit further investigation for use in spinal implants. 

This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 812765 and from the European Union’s Seventh Framework Programme (FP7/2007-2013), grant agreement GA-310477(LifeLongJoints). 

INTRODUCTION 

One of the main limiting factors to the life span of spinal implants is the release of detrimental ions and particles, which are typically produced by wear and corrosion1,2. One suggested approach to overcome these issues is the use of silicon nitride-based coatings on metallic implants because of their low wear rates and their ability to slowly dissolve in aqueous solutions into biocompatible ions only, which could be advantageous in terms of limiting the effects of wear debris and ion release3. A previous study found that alloying the silicon nitride coating with Fe and C did not have a negative effect on mechanical properties nor biocompatibility in a direct contact in vitro test4. However, the dissolution behaviour of the coatings remains to be investigated. Furthermore, due to the close proximity to nerve tissues in spinal implants, the effect of the ions released on the neural tissue is a concern. The present study aimed to study the dissolution behaviour and in vitro neural cell response of SiFeCN coatings. A combinatorial approach was used for efficient screening of different compositions. 

EXPERIMENTAL METHODS 

SiFeCN coatings were deposited on CoCr disc substrates by reactive sputtering in an in-house built equipment, allowing for combinatorial processes, using Si, Fe and C solid targets. Nitrogen was supplied as a reactive gas. The coatings were characterized in 9 points using x-ray photoelectron spectroscopy (XPS), vertical scanning interferometry (VSI) and scanning electron microscopy (SEM). The points were placed in a 3x3 grid with 22.5 mm between each point. 

The dissolution behaviour was evaluated by exposing the coated samples to cell media for 14 days. The obtained extracts were diluted (1:32, 1:48, 1:64 and 1:80 dilution) and used to measure ion levels with inductively coupled plasma (ICP-OES) and to assess indirect biocompatibility in vitro using the MTT assay and glial cells. 

RESULTS AND DISCUSSION 

The XPS results showed compositional gradients of Si ranging between 36.4-47.3 at.%, Fe 1.4-9.3 at.% and C 4.5-13.9 at.% with average surface roughness, Sa, of 7.4 to 11.1 nm, similar to SiN and CoCr reference materials. SEM after exposure displayed signs of dissolution with visibly increased porosity for the coated samples. The SiN reference also showed substantial changes to the surface. The ICP results (Figure 1) showed a reduction in Co ions from the substrate in the coated samples compared to uncoated. Moreover, the addition of Fe and C decreased the ion release from the coating compared to the SiN reference coating. Extract biocompatibility tests suggested that glial cells tolerated the extracts and their dilutions obtained from the coated samples in a dose- dependent manner and the cell viability was comparable to that of the uncoated CoCr and SiN coating. 

CONCLUSIONS 

The findings from this study suggest that using iron and carbon as alloying elements in silicon nitride coatings has the potential to reduce ion release from a metallic substrate and lower the dissolution rate of the coating, while having a comparable cell response to that of the CoCr and SiN control materials. Therefore, SiFeCN coatings merit further investigation as a future option for spinal implants. 

REFERENCES 

1.Shimamura Y. et al., Spine. 33(4):351–355, 2008 2.Vicars R. et al., Comprehensive Biomaterials II. (pp. 246–264), 20173. Pettersson M. et al., ACS Biomaterials Science and Engineering. 2(6):998–1004, 20164. Skjöldebrand C. et al., Materials (Basel). 13(9):1–16, 2020 

ACKNOWLEDGMENTS 

This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 812765 and from the European Union’s Seventh Framework Programme (FP7/2007-2013), grant agreement GA-310477(LifeLongJoints).