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We developed a novel collagen-based biomaterial ink with an integrated antimicrobial peptide (AMP) intended for bone tissue engineering, enabling fabrication of a 3D-printed collagen-scaffold with antibacterial activity. A chemically synthesized derivative of the human AMP KR-12 was designed for enhanced stability and activity against Gram-positive bacteria that are key pathogens in fracture-related infections. KR-12 was covalently conjugated to collagen using both non-selective and maleimide-based coupling strategies. The choice of conjugation method significantly influenced peptide incorporation efficiency and functional availability. Among the resulting formulations, the ink in which KR-12 was conjugated with collagen using a short linker (Coll-cysKR12Q5KD9A SL) demonstrated suitable rheological properties for extrusion-based 3D printing and reduced the growth of Staphylococcus aureus in surrounding culture media. Additionally, this scaffold demonstrated cytocompatibility with primary human osteoblasts (hOBs) in indirect assays. All collagen-KR-12 hydrogels were successfully printed, with Coll-cysKR12-Q5KD9A SL enabling the finest structural resolution. These findings provide an initial proof-of-concept for peptide-functionalized collagen scaffolds fabricated by direct ink writing. However, further studies are required to evaluate scaffold-associated antibacterial activity, biofilm inhibition, and direct osteogenic performance.

In this study, we investigated the mechanical and osteoconductive properties of a novel cross-linked collagen-hydroxyapatite (Coll-HA-XL) biomaterial ink for 3D-printed scaffolds in bone tissue engineering. The biomaterial ink was developed through three stages: initially as a hydrogel, then molded and freeze-dried into disk-shaped forms, and finally as 3D-printed scaffolds subjected to freeze-drying. To optimize the ink, we systematically varied the hydroxyapatite (HA) proportions and the sequence of HA incorporation and collagen cross-linking. The introduction of HA and subsequent collagen cross-linking significantly increased the storage modulus of the hydrogels and enhanced the compressive strength and thermal stability of the freeze-dried 3D-printed scaffolds. These improved mechanical properties enabled the fabrication of osteoconductive scaffolds via direct ink writing. To evaluate biological performance, the 3D-printed Coll-HA-XL scaffolds were seeded with primary human osteoblasts (hOBs). After four weeks of culture, hOBs demonstrated high alkaline phosphatase (ALP) activity and positive osteocalcin (OCN) staining, indicating robust osteoblastic differentiation. These findings highlight the potential of the Coll-HA-XL ink for creating 3D-printed scaffolds tailored for bone tissue engineering applications. 

Spinal implants have been successfully used to treat various spine conditions. However, concerns remain regarding the release of ions and particles that can cause adverse host responses. Moreover, the biological response from cells of the central nervous system to implant wear particles is not well documented. This research studied the interaction of silicon nitride, cobalt oxide, or chromium oxide particles with relevant glial cells, astrocytes and microglia, to assess their effects on viability and activation using a range of relevant concentrations. Astrocyte viability was not impaired by the particles, and expression levels of the astrocyte reactivity markers GFAP and vimentin were unaffected following 24 h of exposure. Moreover, the particles did not alter microglial phagocytic activity, or stimulate the release of the inflammatory cytokines TNF-α and IL-6. Together, these results suggest that the release of such particles from a spinal implant would not induce an inflammatory response in surrounding glial cells. Interestingly, high concentrations of SiN particles reduced microglial cell viability, potentially due to their tendency to form large agglomerates and/or their high dissolution rate. The present work highlights the importance of studying the potential effects of SiN particles on glial cells and encourages further investigations to fully understand the safety aspects of the use of SiN in spinal implants.

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.