| 1. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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A microfluidics-based method for culturing osteoblasts on biomimetic hydroxyapatite
- 2021
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Ingår i: Acta Biomaterialia. - : Elsevier. - 1742-7061 .- 1878-7568. ; 127, s. 327-337
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Tidskriftsartikel (refereegranskat)abstract
- The reliability of conventional cell culture studies to evaluate biomaterials is often questioned, as in vitro outcomes may contradict results obtained through in vivo assays. Microfluidics technology has the potential to reproduce complex physiological conditions by allowing for fine control of microscale features such as cell confinement and flow rate. Having a continuous flow during cell culture is especially advantageous for bioactive biomaterials such as calcium-deficient hydroxyapatite (HA), which may otherwise alter medium composition and jeopardize cell viability, potentially producing false negative results in vitro. In this work, HA was integrated into a microfluidics-based platform (HA-on-chip) and the effect of varied flow rates (2, 8 and 14 µl/min, corresponding to 0.002, 0.008 and 0.014 dyn/cm2, respectively) was evaluated. A HA sample placed in a well plate (HA-static) was included as a control. While substantial calcium depletion and phosphate release occurred in static conditions, the concentration of ions in HA-on-chip samples remained similar to those of fresh medium, particularly at higher flow rates. Pre-osteoblast-like cells (MC3T3-E1) exhibited a significantly higher degree of proliferation on HA-on-chip (8 μl/min flow rate) as compared to HA-static. However, cell differentiation, analysed by alkaline phosphatase (ALP) activity, showed low values in both conditions. This study indicates that cells respond differently when cultured on HA under flow compared to static conditions, which indicates the need for more physiologically relevant methods to increase the predictive value of in vitro studies used to evaluate biomaterials.
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| 2. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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A Universal Microfluidic Platform for In Vitro Biomaterial Evaluation
- 2022
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- INTRODUCTION: Conventionally, the biological properties of biomaterials are evaluated using well plates. Although being a standardized method, it is static in terms of fluid flow and is far from the physiological conditions found in vivo. This work presents a versatile microfluidic system that allows for integration of different biomaterials (ceramic, metals and polymers) under dynamic conditions.METHODS: The Universal Biomaterial-on-Chip (UBOC) consisted of two separate 3D printed (Polylactic acid, Ultimaker 2+) structures: the upper layer which contains the channel through which medium can flow (Fig1A) and the bottom layer that holds and secures the biomaterial in place (Fig 1B). A glass coverslip was taped to the upper layer to tightly seal the channel. Subsequently, an oval Polydimethylsiloxane (PDMS) gasket (l=10mm,w=7mm, h=0.8mm) was inserted into the periphery of the channel in the upper layer. Furthermore, magnets (Ø=12mm, h=3mm) were glued on both sides of the bottom layer. To close the channel, two magnets were placed on the upper layer, causing attraction to the magnets in the bottom layer. The gasket would then directly interface with the biomaterial inside the bottom layer, creating a leak-free channel on its surface. MC3T3-E1 pre-osteoblasts were seeded in the UBOC platform (50,000 cells/cm2) on calcium-deficient hydroxyapatite (HA) (Ø=15mm) and clinical grade titanium (Ti) (Ø=12mm). The cells were cultured for a period of 5 days at a flow rate of 2 μl/min using supplemented MEM-α medium (Hyclone, 10% FBS, 1% Pen-Strep). On day 5, the cells were stained on-chip with Live/Dead stain (Calcein, Propidium Iodide and Hoechst) and subsequently imaged.RESULTS: HA and Ti samples were successfully integrated into the UBOC. Cells cultured on-chip displayed a high degree of viability and confluence on day 5 of culture on both HA and Ti substrates (Fig 2).DISCUSSION & CONCLUSIONS: UBOC presents the possibility for flexible in vitro biomaterial analysis as it allows for easy incorporation of flow to conventional cell culture regimes in a low-cost manner. Via this method,cells can be cultured on the biomaterial with exposure to fluid flow and controlled shear-stress. The platform is compatible with standard characterization methods, such as imaging and biochemical cell analysis. In addition, since the system is designed to be opened and closed, the biomaterial could be easily accessed, harvested and transferred to a regular tissue culture vessel,enabling standard off-chip biochemical assays and protocols to be performed for further analysis.
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| 3. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Bone Cement Embedded in a Microfluidic Device
- 2018
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Konferensbidrag (refereegranskat)abstract
- Calcium phosphate cements (CPCs) have a great potential in the treatment of bone disorders due to their excellent biocompatibility. Although CPCs are promising when implanted in vivo, there is poor correlation between in vitro and in vivo studies. This could be because most conventional in vitro systems lack a 3D architecture, or dynamic conditions (i.e. a continuous refreshment stream). The aim of this work is to embed CPCs into a microfluidic system and evaluate ion and protein exchange at different flow rates.
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| 4. |
- Atif, Abdul Raouf, 1996-
(författare)
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Evaluation of Biological Biomaterial Properties using Microfluidic Systems
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite increased orthopedic biomaterial research activity over previous decades, relatively few novel biomaterials have made it to clinical use. This may partially be due to the inability of existing in vitro testing routines to sufficiently replicate the physiological environment, leading to potentially inaccurate assessments of a biomaterial’s therapeutic potential. To address this, mathematical modelling and microfluidic design principles were assessed as possible supportive strategies to better improve the informativity of in vitro testing approaches.Using principles of the Langmuir isotherm, a predictive computational model was constructed to capture the dynamics of protein and cell adhesion on a biomaterial surface, specifically on calcium-deficient hydroxyapatite, which is a synthetic biomaterial that is compositionally similar to the inorganic phase of the bone. The results demonstrated the success of the model at capturing the trends of the data, thereby indicating potential use as a predicative tool to assist with in vitro data interpretation.Furthermore, attempts were made to improve the in vitro environment towards better physiological relevancy via the introduction of microfluidics, which is method of precise fluid control in micron-sized channels. For instance, the use of microfluidics allows for cell culture under more tissue relevant length scales, as well as the provision of a continuous media flow, which facilitates nutrient delivery and activation of mechanosensitive pathways through shear stress. Through development of such “Biomaterial-on-chip” microfluidic platforms, a general increase in cell viability and proliferation was seen when cells were cultured under flow. The effect of flow on other parameters such as material-induced ionic exchange, immunogenicity and mechanotransduction was also tested using the platform. By the culmination of the thesis work, the Biomaterial-on-chip platform was designed with inherent standardization, allowing for the in vitro testing of different biomaterials of varying shapes and properties under the same conditions in the same platform. All in all, the main conclusion from this thesis work is that cell response can largely differ depending on the chosen culture conditions, which therefore necessities careful consideration of environmental parameters prior to the start of an in vitro biomaterial evaluation study.
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| 5. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Evaluation of Ionic Interactions of Bone Cement-on-Chip
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- INTRODUCTION: Biomaterials are synthetic materials that can be incorporated into the body to replace an impaired physiological function. Apatite calcium phosphate cements (CPCs), used for bone regeneration, give calcium-deficient hydroxyapatite (CDHA) as an end-product after a dissolution-precipitation reaction during fabrication. CDHA has a tendency to uptake calcium and release phosphate into cell culture medium. Potentially, this leads to depletion of calcium ions in solution, which can be detrimental to cell survival. The aim of this work is to embed CDHA in a microfluidic system and evaluate ion exchange at different flow rates.METHODS: CPC paste was cast into a 0.8mm pocket within a Polydimethylsiloxane (PDMS, cured at 60°C for 2h) mould. CPCs were set in 0.9% w/v NaCl at 37°C for 10 days resulting in CDHA. The PDMS containing the CDHA was then bonded to glass, leaving a 0.5mm channel gap. Minimum Essential Media (MEM, 1ml) was pumped through the channel at low (2µl/min), medium (8µl/min) and high (14µl/min) flow rates. A CDHA disc (ø=15mm, h=2mm) was immersed in MEM (1ml) at static conditions (0µl/min) for 24h. Stock Media was taken as control. Calcium and phosphorus concentrations were analysed using Inductively Coupled Plasma Optical Emission Spectroscopy.RESULTS & CONCLUSIONS: CDHA was successfully embedded in a microfluidic chip (Fig. 1A). Observed [Ca] and [P] levels were closer to levels in stock MEM at higher flow rates (Fig. 1B). We anticipate that osteoblast viability will improve when grown under flow, as opposed to static conditions, due to continuous replenishment of cell medium.
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| 6. |
- Atif, Abdul-Raouf, 1996-, et al.
(författare)
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Experimental Characterization and Mathematical Modeling of the Adsorption of Proteins and Cells on Biomimetic Hydroxyapatite
- 2022
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Ingår i: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 7:1, s. 908-920
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Tidskriftsartikel (refereegranskat)abstract
- Biomaterial development is a long process consisting of multiple stages of design and evaluation within the context of both in vitro and in vivo testing. To streamline this process, mathematical and computational modeling displays potential as a tool for rapid biomaterial characterization, enabling the prediction of optimal physicochemical parameters. In this work, a Langmuir isotherm-based model was used to describe protein and cell adhesion on a biomimetic hydroxyapatite surface, both independently and in a one-way coupled system. The results indicated that increased protein surface coverage leads to improved cell adhesion and spread, with maximal protein coverage occurring within 48 h. In addition, the Langmuir model displayed a good fit with the experimental data. Overall, computational modeling is an exciting avenue that may lead to savings in terms of time and cost during the biomaterial development process.
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| 7. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Influence of flow in the adhesion and proliferation of cells on hydroxyapatite integrated in a microscale culture
- 2021
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- INTRODUCTION: Synthetic biomaterials, such as calcium phosphate cements (CPCs), are a promising alternative to autologous bone to enhance bone regeneration. Calcium-deficient hydroxyapatite (CDHA), the end-product of apatite cements, matches the inorganic phase of the bone and exhibits excellent biocompatibility in vivo [1]. However, in vitro, CDHA uptakes calcium ions (Ca2+) from cell culture medium [2], causing detrimental effects on cell activity and function [3]. The aim of this work was to integrate CDHA into a microfluidic chip that provides continued culture medium supply, and to evaluate cell adhesion and proliferation as compared to standard well plates.METHODS:CDHA was integrated in a polydimethylsiloxane (PDMS)-glass microfluidic chip (CDHA-on-chip). PDMS was cured in a 3D-printed mould at 60°C for 2h. α-tricalcium phosphate was mixed with 2.5% w/v Na2HPO4(aq) (liquid-to-powder of 0.65 ml/g) and the CPC was cast within a PDMS pocket. The CPC was immersed in an aqueous solution at 37°C for 10 days to ensure full transformation to CDHA. Through plasma treatment, a glass slide was bonded to the PDMS holding the CDHA, thus forming a 0.5mm channel above the CDHA. CDHA samples were pre-incubated for 24h in minimum essential media (MEM) supplemented with 10% FBS and 1% penicillin-streptomycin (sMEM). Pre-osteoblasts (MC3T3-E1) were seeded at 50,000 cells/cm2 and after a cell adhesion period of 2h, flow was applied for 72h through the chip at different rates: 2, 8 and 14 μl/min. A static (0 μl/min) chip condition was included, where sMEM was manually replaced every 24h. CDHA discs (⌀=6mm, h=2mm) placed in a 96-well plate were used as a standard static control (200 μl sMEM replaced every 24h). At 6h and 72h, the cells were stained with a calcein, propidium iodide and Hoechst triple-stain to assess their adhesion and proliferation, respectively. In a separate experiment, sMEM was flown through the chips for 24h at the aforementioned flow rates, and Ca2+ concentration was quantified via inductively coupled plasma-optical emission spectroscopy (ICP-OES). As control, sMEM in contact with CDHA discs for 24h was evaluated.RESULTS:A larger number of cells adhered on the CDHA-on-chip under flow as opposed to both static CDHA-on-chip and CDHA disc in a well plate. Differences in cell adhesion between the flow conditions were negligible. Cell proliferation at 72h was significantly increased under flow compared to CDHA disc samples (Fig.1A). Static CDHA-on-chip showed almost no viable cells. 2 and 8 μl/min flow conditions showed the greatest cell counts, followed by the 14 μl/min flow condition. At higher flow rates, Ca2+ concentrations were closer to in fresh medium (Fig.1B)DISCUSSION & CONCLUSIONS:The static CDHA-on-chip and disc samples displayed a low degree of cell adhesion and proliferation, which seemed to indicate that ionic exchange led to detrimental cell behaviour. Cells displayed the greatest degree of adhesion and proliferation at a flow rate of 2 and 8 μl/min, probably due to more optimal Ca2+ concentrations. At 14 μl/min, the degree of cell adhesion and proliferation decreased, which could be ascribed to adverse effects of shear stress.
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| 8. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Modelling adsorption of proteins and cells on biomimetic hydroxyapatite
- 2021
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Calcium-deficient hydroxyapatite (CDHA), a biomaterial similar to the inorganic bone matrix, can be used in non-load bearing areas to promote bone regeneration. Upon implantation, CDHA is exposed to blood, leading to serum protein deposition on the surface and enabling cell attachment via membrane-bound receptors. In cell culture studies, biomaterials are often pre-incubated in serum supplemented medium to mimic this process. In this work, to study the extent the protein layer assists in cell adhesion, a Langmuir isotherm-based model for protein and cell adhesion kinetics was used.
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| 9. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Quantitative evaluation of osteoblast proliferation and differentiation on a biomaterial in a microfluidic device
- 2020
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- IntroductionCalcium phosphate cements (CPCs) are able to transform into calcium deficient hydroxyapatite (CDHA), whose crystal size and chemistry closely matches that of the inorganic phase of bone [1]. CDHA readily uptakes calcium ions, and releases phosphate, when immersed in synthetic solutions that mimic physiological fluids [1]. While CPCs are able to enhance bone regeneration in defect sites located in non-load bearing areas, the ionic imbalance that arises from dissolution may also have detrimental effects on cell behavior and function. The purpose of this study was to culture cells on CDHA embedded in a microfluidic chip, under flow, to sustain optimal ionic concentrations, and subsequently evaluate cell proliferation and differentiation. MethodsCPC was cast into a polydimethylsiloxane (PDMS) pocket (h = 0.8 mm) and then set in a 0.9 % NaCl(aq) solution at 37°C for 10 days leading to conversion into CDHA. The CDHA embedded in PDMS were dried and bonded to glass via oxygen plasma treatment, resulting in chips with a 0.5 mm deep channel above the CDHA. In parallel, CDHA discs (⌀ = 6 mm) were set in Teflon molds for the same period of time. The CDHA chips and discs were sterilized with ethanol and pre-incubated with cell culture media overnight. MC3T3-E1 pre-osteoblasts (50,000 cells/cm2) were seeded on the CDHA, and allowed to adhere for 2 h, before initiating a flow of 8 µl/min. Cell proliferation (indirectly measured as the cytosolic lactate dehydrogenase (LDH) enzyme of cells previously adhered to the material) and cell differentiation (alkaline phosphatase activity normalized by total amount of protein) were quantified on day 1, 5 and 10. On day 10, cells were stained with Calcein, Propidium iodide (live/dead assay) and Hoechst (nucleus), and were imaged via fluorescence microscopy. ResultsThe fabrication of the CDHA-on-chip was successful (Fig 1A). There was a faster increase of osteoblast growth on the CDHA-on-chip (under flow) than on discs (static conditions). Specifically, between day 5 and 10, cell number on-chip increased a two-fold as compared to the insignificant change on discs (Fig 1B). Cells on-chip were observed confluent at day 10 (Fig 1C) and seemed to differentiate over time (not shown).ConclusionThe integrated hydroxyapatite platform is a potential alternative for standard in vitro analysis using well plates. Application of flow ameliorates media ionic imbalance, while also providing fresh nutrients and removing waste.
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| 10. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Universal Biomaterial-on-Chip : a versatile platform for evaluating cellular responses on diverse biomaterial substrates
- 2024
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Ingår i: Journal of materials science. Materials in medicine. - : Springer. - 0957-4530 .- 1573-4838. ; 35
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Tidskriftsartikel (refereegranskat)abstract
- Microfluidics has emerged as a promising approach for assessing cellular behavior in vitro, providing more physiologically relevant cell culture environments with dynamic flow and shear stresses. This study introduces the Universal Biomaterial-on-Chip (UBoC) device, which enables the evaluation of cell response on diverse biomaterial substrates in a 3D-printed microfluidic device. The UBoC platform offers mechanical stimulation of the cells and monitoring of their response on diverse biomaterials, enabling qualitative and quantitative in vitro analysis both on- and off-chip. Cell adhesion and proliferation were assessed to evaluate the biocompatibility of materials with different physical properties, while mechanical stimulation was performed to investigate shear-dependent calcium signaling in pre-osteoblasts. Moreover, the applicability of the UBoC platform in creating more complex in vitro models by culturing multiple cell types was demonstrated, establishing a dynamic multicellular environment to investigate cellular interfaces and their significance in biological processes. Overall, the UBoC presents an adaptable tool for in vitro evaluation of cellular behavior, offering opportunities for studying various biomaterials and cell interactions in microfluidic environments.
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| 11. |
- Atif, Abdul Raouf, 1996-, et al.
(författare)
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Universal Biomaterial-on-Chip: A modular platform for flexible biomaterial integration and versatile quantitative assessment
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A requirement for clinical approval is verification of a biomaterial’s functionality and biocompatibility. However, discrepancies between in vitro and in vivo evaluations have been reported, possibly due in part to a lack of physiological relevance of typical in vitro culture set-ups. We introduce a Universal Biomaterial-on-Chip (UBoC), which is a microfluidics device that allows integration of biomaterials with varied shapes and properties and subsequent evaluation of in vitro performance under design considerations that resemble physiological conditions. In addition, UBoC operates with multifunctional modalities such as continuous perfusion, shear stress mechanostimulation and cell co-culture. The device is constructed using simple 3D printing and microfabrication techniques and its cell culture area resembles a 96-well plate (0.32 cm2). Successful cell adhesion and proliferation was observed on-chip on different materials (hydroxyapatite, titanium and fibrin) using fluorescence microscopy. Furthermore, device applicability for mechanostimulation was demonstrated through shear stimulation, where sensitivity of pre-osteoblasts to flow was captured via live Ca2+ imaging. Finally, the modularity of the UBoC platform for on-chip co-culture experiments was established after simple modifications of on-board fluidic arrangements. Overall, the UBoC presents a useful tool that augments existing in vitro testing strategies and enables thorough comparisons between biomaterials in tunable culture conditions.
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| 12. |
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| 13. |
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| 14. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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A microfluidic-based approach to investigate the inflammatory response of macrophages to pristine and drug-loaded nanostructured hydroxyapatite
- 2022
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Ingår i: Materials Today Bio. - : Elsevier. - 2590-0064. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.
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| 15. |
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| 16. |
- Carter, Sarah-Sophia D., 1994-, et al.
(författare)
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The effect of unidirectional and recirculating flow on the behavior of MC3T3-E1 preosteoblast-like cells in a microfluidic system
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Microfluidic systems have been proposed as a promising tool to capture enhanced physiological relevance in an in vitro setting. Although offering new opportunities, maintaining cells in such systems differs significantly from culturing cells under conventional static in vitro conditions. In order to directly compare the results from these two set-ups and to make more conclusive statements regarding the differences between them, it is important to carefully consider which factors can affect cell behavior. In this work, we investigated the effect of the flow type, namely unidirectional and recirculating flow, on the behavior of MC3T3-E1 preosteoblast-like cells and compared this to cells cultured under standard static cell culture conditions. Cell proliferation and differentiation (i.e. alkaline phosphatase activity, extracellular collagen and mineral matrix deposition) were overall higher for the cells maintained under static conditions when compared to either of the microfluidic set-ups. It should however be noted that cell proliferation showed to be highly dependent on the frequency of medium renewal and the amount of medium used in the static conditions. In addition to that, we demonstrated that the use of differentiation medium resulted in higher proliferation than regular growth medium, regardless the culture system used. No clear differences in cell proliferation and differentiation were found between the cells exposed to unidirectional or recirculating flow. Interestingly, secreted IGF-I was higher in the microfluidic systems, where unidirectional flow seemed to enhance the secretion. Overall, our results demonstrated that in vitro cell culture conditions can drastically affect cell response and should therefore be carefully considered.
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| 17. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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Exploring microfluidics as a tool to evaluate the biological properties of a titanium alloy under dynamic conditions
- 2020
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Ingår i: Biomaterials Science. - : Royal Society of Chemistry (RSC). - 2047-4830 .- 2047-4849. ; 8, s. 6309-6321
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Tidskriftsartikel (refereegranskat)abstract
- To bring novel biomaterials to clinical use, reliable in vitro models are imperative. The aim of this work was to develop a microfluidic tool to evaluate the biological properties of biomaterials for bone repair. Two approaches to embed medical grade titanium (Ti6Al4V) on-chip were explored. The first approach consisted of a polydimethylsiloxane microfluidic channel placed onto a titanium disc, held together by an additively manufactured fixture. In the second approach, a titanium disc was assembled onto a microscopic glass slide, using a double-sided tape microfluidic channel. Both approaches demonstrated potential for maintaining MC3T3-E1 preosteoblast-like cell cultures on-chip, as was shown by the vast majority of living cells after 1 day. In addition, the cells cultured on-chip showed a more elongated morphology compared to cells grown under static conditions and a tendency to align to the direction of the flow. For longer-term (i.e. 10 days) studies, the glass-based chip was selected. Assessment of cell viability showed a high number of living cells during the entire experimental period. Cell proliferation and differentiation studies revealed an increase in cell proliferation on-chip, suggesting that proliferation was the dominating process at the detriment of differentiation in this micrometric dynamic environment. These results illustrate the importance of optimizing in vitro cell culture conditions and how these may affect biomaterial testing outcomes. Overall, this work provides a step towards more in vivo-like microfluidic testing platforms, which are expected to provide more reliable in vitro screening of biomaterials.
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| 18. |
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| 19. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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Medical grade titanium on-chip : assessing the biological properties of biomaterials for bone regeneration
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Medical grade titanium on-chip: assessing the biological properties of biomaterials for bone regeneration Sarah-Sophia D. Carter1, Hugo Nguyen2, Milena Moreira1, Maria Tenje1, and Gemma Mestres11Department of Engineering Sciences, Science for Life Laboratory, Uppsala University, Sweden2Department of Engineering Sciences, Uppsala University, Sweden IntroductionBefore entering the clinic, biomaterials need to be thoroughly evaluated, which requires accurate in vitro models. In this work, we have developed a microfluidic device that could be used to assess the biological properties of biomaterials, in a more in vivo-like environment than what is currently possible. MethodsOur device consists of a polydimethylsiloxane (PDMS, Sylgard 184) microfluidic channel (l= 6 mm, w= 2 mm, h= 200 µm) and a titanium disc (Ti6Al4V, at bottom), held together by an additively manufactured fixture (Fig. 1A). PDMS was cured overnight at 65°C on a silicon wafer master. Once the microchannel and titanium disc were positioned, MC3T3-E1 pre-osteoblast-like cells were seeded (50,000 cells/cm2). After 5 hours incubation under standard culture conditions, flow was started (2 μl/min). As a control, MC3T3-E1 cells were seeded onto plain titanium discs off-chip. Cell viability and morphology were assessed after 20 hours by calcein-AM/propidium iodide (PI), staining live and dead cells respectively. Results and discussionFigure 1B and 1C show calcein-AM/PI stained MC3T3-E1 cells cultured on-chip and figure 1D shows the control, MC3T3-E1 cells cultured off-chip. The potential to culture cells in our chip was confirmed by the presence of a majority of viable cells (green) with a similar morphology as the control sample. The reason for the increased amount of dead cells (red) on-chip compared to the control needs to be further examined, which requires longer-term experiments.ConclusionWe have set the first steps towards a microfluidic tool for the assessment of biological properties of biomaterials.
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| 20. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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On-chip evaluation of the biological properties of medical-grade titanium
- 2020
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Konferensbidrag (refereegranskat)abstract
- On-chip evaluation of the biological properties of medical-grade titaniumSarah-Sophia D. Carter1, Laurent Barbe1, Maria Tenje1 and Gemma Mestres1,*1 Department of Materials Science and Engineering, Science for Life Laboratory, Uppsala University, Uppsala, Sweden*E-mail: gemma.mestres@angstrom.uu.seIntroductionBefore entering the clinic, biomaterials need to be thoroughly evaluated, which requires accurate in vitro models. However, it has been shown that the currently used models correlate poorly with in vivo results [1]. In this work, microfluidic chips that integrate medical grade titanium (Ti6Al4V) were fabricated and subsequently used to study the biological properties of this biomaterial on-chip. The overall goal of this project is to develop on-chip platforms to evaluate novel biomaterials for bone regeneration.Theory and Experimental procedureA glass coverslip (175 µm thick) was laser cut to fit a Ti6Al4V disc (⌀ = 12 mm) and assembled onto a microscopic glass slide (1 mm thick) using double-sided tape (140 µm thick), the latter shaping the microfluidic channel. To ensure a tight seal between the glass coverslip and the Ti6Al4V disc, a UV adhesive was used. MC3T3-E1 pre-osteoblast cells were seeded at 45,000 cells/cm2 and allowed to adhere for 4 hours prior to starting the perfusion. After 1, 5 and 10 days, cell proliferation and cell differentiation were evaluated by the lactate dehydrogenase (LDH) assay (used as an indirect method to quantify the cytosolic enzyme LDH of cells previously adhered to the biomaterial) and the alkaline phosphatase (ALP) assay. As a static control, MC3T3-E1 cells were seeded on Ti6Al4V discs in a well plate.Results and DiscussionMC3T3-E1 cells were successfully grown on Ti6Al4V on-chip, as was confirmed by an increase in cell proliferation over time, which became significantly elevated compared to the static condition from day 5 onwards (Figure 1A). Cell differentiation increased over the studied period for both on-chip and static conditions (Figure 1B). However, in the static condition, ALP activity reached much higher levels compared to on-chip. All together, these results correlate well with the fact that cell proliferation is the dominating process on-chip during the experimental period.ConclusionMicrofluidic chips that integrate medical grade Ti6Al4V were fabricated and used to evaluate the biological properties of this biomaterial under dynamic conditions. Cell proliferation and differentiation studies indicate that MC3T3-E1 cells cultured on Ti6Al4V on-chip remain in a proliferative state during the time period of 10 days.AcknowledgmentsGM acknowledges Formas, Vetenskapsrådet and Göran Gustafsson´s Foundation for financial support.References[1] G. Hulsart-Billström et al., European Cells and Materials 31, 312-322 (2016).
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| 21. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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PDMS leaching and its implications for on-chip studies focusing on bone regeneration applications
- 2020
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Ingår i: Organs-on-a-Chip. - : Elsevier. - 2666-1020. ; 2
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Tidskriftsartikel (refereegranskat)abstract
- Polydimethylsiloxane (PDMS) is among the most widely used materials for organ-on-chip systems. Despite itsmultiple beneficial characteristics from an engineering point of view, there is a concern about the effect of PDMSon the cells cultured in such devices. The aim of this study was to enhance the understanding of the effect of PDMSon cellular behavior in a context relevant for on-chip studies. The focus was put on an indirect effect of PDMS,namely leaching of uncrosslinked oligomers, particularly for bone regeneration applications. PDMS-based chipswere prepared and analyzed for the potential release of PDMS oligomers within the microfluidic channel whenkept at different flow rates. Leaching of uncrosslinked oligomers from PDMS was quantified as silicon concen-tration by inductively coupled plasma - optical emission spectrometry and further confirmed by mass spec-trometry. Subsequently, PDMS-leached media, with a silicon concentration matching the on-chip experiment,were prepared to study cell proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts and humanmesenchymal stem cells. The silicon concentration initially detected in the media was inversely proportional tothe tested flow rates and decreased to control levels within 52 h. In addition, by curing the material overnightinstead of 2 h, regardless of the curing temperature (65 and 80 C), a large reduction in silicon concentration wasfound, indicating the importance of the PDMS curing parameters. Furthermore, it was shown that PDMS oligo-mers enhanced the differentiation of MC3T3-E1 pre-osteoblasts, this being a cell type dependent effect as nochanges in cell differentiation were observed for human mesenchymal stem cells. Overall, this study illustrates theimportance of optimization steps when using PDMS devices for biological studies, in particular PDMS curingconditions and extensive washing steps prior to an experiment.
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| 22. |
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| 23. |
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| 24. |
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| 25. |
- D’Elía, Noelia L., et al.
(författare)
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Development and characterisation of bilayered periosteum-inspired composite membranes based on sodium alginate-hydroxyapatite nanoparticles
- 2020
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier. - 0021-9797 .- 1095-7103. ; 572, s. 408-420
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Tidskriftsartikel (refereegranskat)abstract
- Background and aim: Membranes for guided bone regeneration should have a mechanical structure and a chemical composition suitable for mimicking biological structures. In this work, we pursue the develop- ment of periosteum-inspired bilayered membranes obtained by crosslinking alginate with different amounts of nanohydroxyapatite.Experiments: Alginate-nanohydroxyapatite interaction was studied by rheology and infrared spectroscopy measurements. The membranes were characterized regarding their tensile strength, degrada- tion and surface morphology. Finally, cell cultures were performed on each side of the membranes.Findings: The ionic bonding between alginate polysaccharide networks and nanohydroxyapatite was proven, and had a clear effect in the strength and microstructure of the hydrogels. Distinct surface charac- teristics were achieved on each side of the membranes, resulting in a highly porous fibrous side and a mineral-rich side with higher roughness and lower porosity. Moreover, the effect of amount of nanohydroxyapatite was reflected in a decrease of the membranes’ plasticity and an increment of degradation rate. Finally, it was proved that osteoblast-like cells proliferated and differentiated on the mineral-rich side, specially when a higher amount of nanohydroxyapatite was used, whereas fibroblasts-like cells were able to proliferate on the fibrous side. These periosteum-inspired membranes are promising biomaterials for guided tissue regeneration applications.
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| 26. |
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| 27. |
- Gallinetti, Sara, 1985-, et al.
(författare)
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A novel strategy to enhance interfacial adhesion in fiber-reinforced calcium phosphate cement
- 2017
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Ingår i: Journal of The Mechanical Behavior of Biomedical Materials. - : Elsevier BV. - 1751-6161 .- 1878-0180. ; 75, s. 495-503
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Tidskriftsartikel (refereegranskat)abstract
- Calcium phosphate cements (CPCs) are extensively used as synthetic bone grafts, but their poor toughness limits their use to non-load-bearing applications. Reinforcement through introduction of fibers and yarns has been evaluated in various studies but always resulted in a decrease in elastic modulus or bending strength when compared to the CPC matrix. The aim of the present work was to improve the interfacial adhesion between fibers and matrix to obtain tougher biocompatible fiber-reinforced calcium phosphate cements (FRCPCs). This was done by adding a polymer solution to the matrix, with chemical affinity to the reinforcing chitosan fibers, namely trimethyl chitosan (TMC). The improved wettability and chemical affinity of the chitosan fibers with the TMC in the liquid phase led to an enhancement of the interfacial adhesion. This resulted in an increase of the work of fracture (several hundred-fold increase), while the elastic modulus and bending strength were maintained similar to the materials without additives. Additionally the TMC-modified CPCs showed suitable biocompatibility with an osteoblastic cell line.
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| 28. |
- Jocic, Simonne, et al.
(författare)
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Fabrication of user-friendly and biomimetic 1,1′-carbonyldiimidazole cross-linked gelatin/agar microfluidic devices
- 2017
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Ingår i: Materials Science and Engineering C. - : Elsevier BV. - 0928-4931 .- 1873-0191. ; 76, s. 1175-1180
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a straightforward technique for fabricating user-friendly and biomimetic microfluidic devices out of a gelatin/agar gel cross-linked with 1,1′-carbonyldiimidazole. The fabrication procedure requires only inexpensive starting materials such as glass capillaries and wires to mold 3D cylindrical channels into the gel with the possibility of achieving channel diameters of 375 μm and 1000 μm. We demonstrate that the channel absent of gel injury can retain fluid within its dimensions for at least 7 h. We also show that the device material does not autofluoresce nor provide hindrances with fluorescent imaging. A discussion of the chemical linkage identities of cross-linked gelatin/agar is included via ATR-FTIR studies. Crosslinking of the gelatin/agar is further confirmed by the lack of a gel to sol transition at physiological temperature as assessed by DSC measurements. SEM micrographs that demonstrate the 100 nm mean pore width of the cross-linked gelatin/agar are provided. This device is considered biomimetic because it represents components present in the natural extracellular matrix such as collagen and proteoglycans in the form of cross-linked gelatin/agar.
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| 29. |
- Kadekar, Sandeep, et al.
(författare)
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Effect of the Addition Frequency of 5-Azacytidine in Both Micro- and Macroscale Cultures
- 2021
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Ingår i: Cellular and Molecular Bioengineering. - : Springer Nature. - 1865-5025 .- 1865-5033. ; 14, s. 121-130
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Human mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. Previous studies have reported that a single addition of 5-azacytidine (5-AzaC) causes the differentiation of hMSCs towards a myocardial lineage. The aim of this work was to evaluate the effect of 5-AzaC addition frequency on hMSCs priming (i.e., indicating an early genetic differentiation) using two culture environments.Methods: hMSCs were supplemented with 5-AzaC while cultured in well plates and in microfluidic chips. The impact of 5-AzaC concentration (10 and 20 mu M) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPAR gamma (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated.Results: Daily delivering 5-AzaC caused a higher upregulation of PPAR gamma, SOX9 and RUNX2 in comparison to a single dose delivery, both under static well plates and dynamic microfluidic cultures. A particularly high gene expression of PPAR gamma (tenfold-change) could indicate priming of hMSCs towards adipocytes.Conclusions: Both macro- and microscale cultures provided results with similar trends, where addition frequency of 5-AzaC was a crucial factor to upregulate several genes. Microfluidics technology was proven to be a suitable platform for the continuous delivery of a drug and could be used for screening purposes in tissue engineering research.
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| 30. |
- Lu, Xi, et al.
(författare)
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Selenium- and tellurium-based antioxidants for modulating inflammation and effects on osteoblastic activity
- 2017
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Ingår i: Antioxidants. - : MDPI AG. - 2076-3921. ; 6:13, s. 1-13
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Tidskriftsartikel (refereegranskat)abstract
- Increased oxidative stress plays a significant role in the etiology of bone diseases. Heightened levels of H2O2 disrupt bone homeostasis, leading to greater bone resorption than bone formation. Organochalcogen compounds could act as free radical trapping agents or glutathione peroxidase mimetics, reducing oxidative stress in inflammatory diseases. In this report, we synthesized and screened a library of organoselenium and organotellurium compounds for hydrogen peroxide scavenging activity, using macrophagic cell lines RAW264.7 and THP-1, as well as human mono- and poly-nuclear cells. These cells were stimulated to release H2O2, using phorbol 12-myristate 13-acetate, with and without organochalogens. Released H2O2 was then measured using a chemiluminescent assay over a period of 2 h. The screening identified an organoselenium compound which scavenged H2O2 more effectively than the vitamin E analog, Trolox. We also found that this organoselenium compound protected MC3T3 cells against H2O2 -induced toxicity, whereas Trolox did not. The organoselenium compound exhibited no cytotoxicity to the cells and had no deleterious effects on cell proliferation, viability, or alkaline phosphatase activity. The rapidity of H2O2 scavenging and protection suggests that the mechanism of protection is due to the direct scavenging of extracellular H2O2. This compound is a promising modulators of inflammation and could potentially treat diseases involving high levels of oxidative stress.
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| 31. |
- Mestres, Gemma, 1984-, et al.
(författare)
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A practical guide for evaluating the osteoimmunomodulatory properties of biomaterials
- 2021
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Ingår i: Acta Biomaterialia. - : Elsevier. - 1742-7061 .- 1878-7568. ; 130, s. 115-137
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Forskningsöversikt (refereegranskat)abstract
- Biomaterials offer a promising approach to repair bone defects. Whereas traditional studies predominantly focused on optimizing the osteogenic capacity of biomaterials, less focus has been on the immune response elicited by them. However, the immune and skeletal systems extensively interact, a concept which is referred to as ‘osteoimmunology’. This realization has fuelled the development of biomaterials with favourable osteoimmunomodulatory (OIM) properties, aiming to modulate the immune response and to support bone regeneration, thereby affecting the success of an implant.Given the plethora of in vitro assays used to evaluate the OIM properties of biomaterials, it may be challenging to select the right methods to produce conclusive results. In this review, we aim to provide a comprehensive and practical guide for researchers interested in studying the OIM properties of biomaterials in vitro. After a concise overview of the concept of osteoimmunology, emphasis is put on the methodologies that are regularly used to evaluate the OIM properties of biomaterials. First, a description of the most commonly used cell types and cell culture media is provided. Second, typical experimental set-ups and their relevant characteristics are discussed. Third, a detailed overview of the generally used methodologies and readouts, including cell type-specific markers and time points of analysis, is given. Finally, we highlight the promise of advanced approaches, namely microarrays, bioreactors and microfluidic-based systems, and the potential that these may offer to the osteoimmunology field.
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| 32. |
- Mestres, Gemma, 1984-, et al.
(författare)
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Advantages of microfluidic systems for studying cell-biomaterial interactions : focus on bone regeneration applications
- 2019
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Ingår i: Biomedical Engineering & Physics Express. - : IOP Publishing. - 2057-1976. ; 5:3
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Forskningsöversikt (refereegranskat)abstract
- The poor correlation between in vitro and in vivo studies emphasises the lack of a reliable methodology for testing the biological properties of biomaterials in the bone tissue regeneration field. Moreover, the success of clinical trials is not guaranteed even with promising results in vivo. Therefore, there is a need for a more physiologically relevant in vitro model to test the biological properties of biomaterials. Microfluidics, which is a field concerning the manipulation and control of liquids at the submillimetre scale, can use channel geometry, cell confinement and fluid flow to recreate a physiological-like environment. This technology has already proven to be a powerful tool in studying the biological response of cells in defined environments, since chemical and mechanical inputs as well as cross-talk between cells can be finely controlled. Moving a step further in complexity, biomaterials can be integrated into microfluidic systems to evaluate biomaterial-cell interactions. The biomaterial- microfluidics combination has the potential to produce more physiologically relevant models to better screen the biological interactions established between biomaterials and cells. This review is divided into two main sections. First, several possible cell-based assays for bone regeneration studies in microfluidic systems are discussed. Second, and the ultimate goal of the review, is to discuss how the gap between in vitro and in vivo studies can be shortened by bridging the biomaterials and microfluidics fields.
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| 33. |
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| 34. |
- Mestres, Gemma, 1984-, et al.
(författare)
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In vivo efficiency of antimicrobial inorganic bone grafts in osteomyelitis Treatments
- 2019
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Ingår i: Materials science & engineering. C, biomimetic materials, sensors and systems. - : Elsevier BV. - 0928-4931 .- 1873-0191. ; 97, s. 84-95
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Tidskriftsartikel (refereegranskat)abstract
- The purpose of the present work was to evaluate in vivo different antimicrobial therapies to eradicate osteomyelitis created in the femoral head of New Zealand rabbits. Five phosphate-based cements were evaluated: calcium phosphate cements (CPC) and calcium phosphate foams (CPF), both in their pristine form and loaded with doxycycline hyclate, and an intrinsic antimicrobial magnesium phosphate cement (MPC; not loaded with an antibiotic). The cements were implanted in a bone previously infected with Staphylococcus aureus to discern the effects of the type of antibiotic administration (systemic vs. local), porosity (microporosity, i.e. <5 μm vs. macroporosity, i.e. >5 μm) and type of antimicrobial mechanism (release of antibiotic vs. intrinsic antimicrobial activity) on the improvement of the health state of the infected animals. A new method was developed, with a more comprehensive composite score that integrates 5 parameters of bone infection, 4 parameters of bone structural integrity and 4 parameters of bone regeneration. This method was used to evaluate the health state of the infected animals, both before and after osteomyelitis treatment. The results showed that the composite score allows to discern statistically significant differences between treatments that individual evaluations were not able to identify. Despite none of the therapies completely eradicated the infection, it was observed that macroporous materials (CPF and CPFd, the latter loaded with doxycycline hyclate) and intrinsic antimicrobial MPC allowed a better containment of the osteomyelitis. This study provides novel insights to understand the effect of different antimicrobial therapies in vivo, and a promising comprehensive methodology to evaluate the health state of the animals was developed. We expect that the implementation of such methodology could improve the criteria to select a proper antimicrobial therapy.
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| 35. |
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| 36. |
- Mestres, Gemma, 1984-, et al.
(författare)
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Novel magnesium phosphate cements with high early strength and antibacterial properties
- 2011
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Ingår i: Acta Biomaterialia. - : Elsevier BV. - 1742-7061 .- 1878-7568. ; 7, s. 1853-1861
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Tidskriftsartikel (refereegranskat)abstract
- Magnesium phosphate cements (MPCs) have been extensively used as fast setting repair cements in civil engineering. They have properties that are also relevant to biomedical applications, such as fast setting, early strength acquisition and adhesive properties. However, there are some aspects that should be improved before they can be used in the human body, namely their highly exothermic setting reaction and the release of potentially harmful ammonia or ammonium ions. In this paper a new family of MPCs was explored as candidate biomaterials for hard tissue applications. The cements were prepared by mixing magnesium oxide (MgO) with either sodium dihydrogen phosphate (NaH2PO4) or ammonium dihydrogenphosphate (NH4H2PO4), or an equimolar mixture of both. The exothermia and setting kinetics of the new cement formulations were tailored to comply with clinical requirements by adjusting the granularity of the phosphate salt and by using sodium borate as a retardant. The ammonium-containing MPC resulted in struvite (MgNH4PO46H2O) as the major reaction product, whereas the MPC prepared with sodium dihydrogenphosphate resulted in an amorphous product. Unreacted magnesium oxide was found in all the formulations.The MPCs studied showed early compressive strengths substantially higher than that of apatitic calcium phosphate cements. The Na-containing MPCs were shown to have antibacterial activity against Streptococcus sanguinis, which was attributed to the alkaline pH developed during the setting reaction.
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| 37. |
- Motisuke, Mariana, et al.
(författare)
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Influence of Si substitution on the reactivity of α-tricalcium phosphate
- 2017
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Ingår i: Materials science & engineering. C, biomimetic materials, sensors and systems. - : Elsevier BV. - 0928-4931 .- 1873-0191. ; 75, s. 816-821
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Tidskriftsartikel (refereegranskat)abstract
- Silicon substituted calcium phosphates have been widely studied over the last ten years due to their enhanced osteogenic properties. Notwithstanding, the role of silicon on α-TCP reactivity is not clear yet. Therefore, the aim of this work was to evaluate the reactivity and the properties of Si-α-TCP in comparison to α-TCP. Precursor powders have similar properties regarding purity, particle size distribution and specific surface area, which allowed a better comparison of the Si effects on their reactivity and cements properties. Both Si-α-TCP and α-TCP hydrolyzed to a calcium-deficient hydroxyapatite when mixed with water but their conversion rates were different. Si-α-TCP exhibited a slower setting rate than α-TCP, i.e. kSSA for Si-TCP (0.021 g·m− 2·h− 1) was almost four times lower than for α-TCP (0.072 g·m− 2·h− 1). On the other hand, the compressive strength of the CPC resulting from fully reacted Si-α-TCP was significantly higher (12.80 ± 0.38 MPa) than that of α-TCP (11.44 ± 0.54 MPa), due to the smaller size of the entangled precipitated apatite crystals.
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| 38. |
- Singh, Vijay P, et al.
(författare)
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Effect of a Bromo Substituent on the Glutathione Peroxidase Activity of a Pyridoxine-like Diselenide
- 2015
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Ingår i: The Journal of Organic Chemistry. - : American Chemical Society (ACS). - 0022-3263 .- 1520-6904. ; 50:15, s. 7385-7395
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Tidskriftsartikel (refereegranskat)abstract
- In search for better mimics of the glutathioneperoxidase enzymes, pyridoxine-like diselenides 6 and 11,carrying a 6-bromo substituent, were prepared. Reaction of2,6-dibromo-3-pyridinol 5 with sodium diselenide provided 6via aromatic nucleophilic substitution of the 2-bromosubstituent. LiAlH4 caused reduction of all four ester groupsand returned 11 after acidic workup. The X-ray structure of 6showed that the dipyridyl diselenide moiety was kept in analmost planar, transoid conformation. According to NBOanalysis,this was due to weak intramolecular Se···O (1.1 kcal/mol) and Se···N-interactions (2.5 kcal/mol). That the 6-bromo substituent increased the positive charge on seleniumwas confirmed by NPA-analysis and seen in calculated andobserved 77Se NMR-shifts. Diselenide 6 showed a more than 3-fold higher reactivity than the corresponding des-bromocompound 3a and ebselen when evaluated in the coupled reductase assay. Experiments followed for longer time (2 h) confirmedthat diselenide 6 is a better GPx-catalyst than 11. On the basis of 77Se-NMR experiments, a catalytic mechanism for diselenide 6was proposed involving selenol, selenosulfide and seleninic acid intermediates. At low concentration (10 μM) where it showedonly minimal toxicity, it could scavenge ROS produced by MNC- and PMNC-cells more efficiently than Trolox.
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| 39. |
- Widhe, Mona, et al.
(författare)
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Functionalized silk promotes cell migration into calcium phosphate cements by providing macropores and cell adhesion motifs
- 2022
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Ingår i: Ceramics International. - : Elsevier BV. - 0272-8842 .- 1873-3956. ; 48:21, s. 31449-31460
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Tidskriftsartikel (refereegranskat)abstract
- Calcium phosphate cements (CPCs) are attractive synthetic bone grafts as they possess osteoconductive and osteoinductive properties. Their biomimetic synthesis grants them an intrinsic nano-and microporosity that resembles natural bone and is paramount for biological processes such as protein adhesion, which can later enhance cell adhesion. However, a main limitation of CPCs is the lack of macroporosity, which is crucial to allow cell colonization throughout the scaffold. Moreover, CPCs lack specific motifs to guide cell interactions through their membrane proteins. In this study, we explore a strategy targeting simultaneously both macroporosity and cell binding motifs within CPCs by the use of recombinant silk. A silk protein functionalized with the cell binding motif RGD serves as foaming template of CPCs to achieve biomimetic hydroxyapatite (HA) scaffolds with multiscale porosity. The synergies of RGD-motifs in the silk macroporous template and the biomimetic features of HA are explored for their potential to enhance mesenchymal stem cell adhesion, proliferation, migration and differentiation. Macroporous Silk-HA scaffolds improve initial cell adhesion compared to a macroporous HA in the absence of silk, and importantly, the presence of silk greatly enhances cell migration into the scaffold. Additionally, cell proliferation and osteogenic differentiation are achieved in the scaffolds.
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