Self‐Assembly of Structured Colloidal Gels for High‐Resolution 3D Micropatterning of Proteins at Scale

Self‐assembly, the spontaneous ordering of components into patterns, is widespread in nature and fundamental to generating function across length scales. Morphogen gradients in biological development are paradigmatic as both products and effectors of self‐assembly and various attempts have been made to reproduce such gradients in biomaterial design. To date, approaches have typically utilized top‐down fabrication techniques that, while allowing high‐resolution control, are limited by scale and require chemical cross‐linking steps to stabilize morphogen patterns in time. Here, a bottom‐up approach to protein patterning is developed based on a novel binary reaction‐diffusion process where proteins function as diffusive reactants to assemble a nanoclay‐protein composite hydrogel. Using this approach, it is possible to generate scalable and highly stable 3D patterns of target proteins down to sub‐cellular resolution through only physical interactions between clay nanoparticles and the proteins and ions present in blood. Patterned nanoclay gels are able to guide cell behavior to precisely template bone tissue formation in vivo. These results demonstrate the feasibility of stabilizing 3D gradients of biological signals through self‐assembly processes and open up new possibilities for morphogen‐based therapeutic strategies and models of biological development and repair.


Introduction
[3] Recapitulation of spatially controlled presentation of biochemical cues is of value for fundamental DOI: 10.1002/adma.202304461studies of biological development and is likely to be key in the regeneration of functional tissues in medicine.Hydrogels are a promising candidate material in this context since their hydrated polymeric networks can emulate the viscoelastic properties of the ECM and potentially allow for the loading of large quantities of protein. [4]chieving patterning in hydrogels is difficult, however, as it requires overcoming the fluctuations inherent to their high water content.
To date, the traditional drug delivery paradigm of slow-release has dominated attempts to control the presentation of biomolecules by hydrogel scaffolds.However, slow-release strategies are poorly suited to achieving the precisely defined protein gradients at play in natural morphogenesis.More recent approaches to stabilizing high-resolution 3D patterning of proteins in hydrogels have relied on top-down assembly methods such as photopatterning, [5] electrophoresis, [6] 3D printing [7] and microfluidics. [8]While these various approaches have demonstrated their utility for the control of cell functions such as cell adhesion, migration, and differentiation they each face challenges of scaleup, stability, protein activity, and biocompatibility.[11] Furthermore, the in vivo utility of each of these methods remains largely untested.
In biological development, protein gradients are themselves a product of self-assembly processes.[14] However, as yet, the potential to exploit RD for engineering morphogen gradients remains unexplored. [15]In the present study, we demonstrate the utility of a simple binary RD system involving physical interactions between an arrested nanoclay colloidal gel and diffusing proteins to assemble stable patterning of bioactive proteins within a structured protein-clay nanocomposite hydrogel.
The nanoclay used in this study is the synthetic hectorite-LAPONITE (herein Laponite) a lithium magnesium sodium Assembly of 3D protein patterning within nanoclay colloidal gels.a) Laponite clay nanoparticles approximate 25 nm disks of 1 nm thickness and possess a permanent negative surface charge and a weak positive edge charge at neutral pH that forms viscous colloidal dispersions in water above ≈2 wt.%.b) After drop-casting a viscous dispersion of Laponite into a high-concentration protein solution (concentrations in the order of 1-100 mg mL −1 ) and then, after a given time, transferring the gel to a lower concentration solution (concentrations in the order of 1-100 μg mL −1 ) of labeled protein a sharply defined band of labeled protein within the gel was observed.c) For example, 3D reconstruction of confocal image and analysis of transparent gel droplets exposed to 10 mg mL −1 BSA for 1 h and then 100 μg mL −1 FITC BSA for 1 h reveals a sharp band of fluorescent protein patterned between the gel periphery and core (scale bar, 200 μm), the plot presents fluorescent intensity within a linear region of interest (running from top left to the bottom right of the image.d) The system supports the assembly and patterning of structures of a range of size and shapes as defined by the initial casting.For example, UV transilluminator images of i) clay-gel droplets of 20 μL incubated in 60% FCS PBS for 170 min and then in 100 μg mL −1 FITC BSA for 48 h (scale bar, 200 μm); ii) 1 mL clay-gel star-shape assembled in 50 mg mL −1 BSA PBS for 4 h and patterned with 500 μg mL −1 FITC BSA for 24 h; iii) a segmented 1 mL clay-gel cylinder assembled in 100% FCS for 72 h and patterned with 250 μg mL −1 FITC BSA for 96 h (scale bars, 4 mm).Similar results were obtained for n = 3 independent samples.silicate (Na 0.7 + [(Si 8 Mg 5.5 Li 0.3 )O 20 (OH) 4 ] −0.7 ) consisting of rigid disk-shaped particles of ≈25 nm diameter and 1 nm thickness (Figure 1a).Owing to its chemical structure it possesses a negative face charge and a weak positive rim charge at neutral pH. [16]aponite particles form delaminated dispersions in water via osmotic swelling and generate unique rheological properties due to the charge anisotropy of the particles that are subject to a range of mutual repulsive (face-to-face, edge-edge) and attractive (face-to-edge) electrostatic interactions. [17]These interactions and the resultant structure and properties of the colloid are highly sensitive to changes in ionic concentration, pH, and the presence of organic compounds. [18]We have previously described the formation of stable, transparent protein-clay gels following the introduction of a viscous arrested nanoclay colloid into blood serum.We have also demonstrated the utility of such gels to bind and deliver growth factors to sustain localized concentrations in vivo to initiate regenerative responses. [19,20]Here we characterize Laponite gelation in proteins as an RD process and demonstrate how control over this process allows for stable protein patterning.

Results
Assembly of 3D protein patterning in nanoclay gels was observed upon casting a viscous clay-gel suspension into a concentrated protein solution (such as blood serum or bovine serum albumin (BSA)) before transfer to a lower concentration solution of fluorescently labeled protein (e.g., FITC-BSA, in the order of 10-100 μg mL −1 ) (Figure 1b).This resulted in a transparent gel, of dimensions and shape controlled by the initial casting, containing a sharply defined band of fluorescent protein arrested between the gel periphery and its core (Figure 1b-d; Figure S1, Supporting Information).Once assembled, patterning was found to be unusually stable, with gels immersed in a saline solution displaying negligible change in the distribution or fluorescent intensity of the labeled protein even after several years of storage (Figure S2, Supporting Information).
Subsequent confocal studies where FITC-BSA was loaded without, concurrent with, or (as above) after assembly in highconcentration BSA, revealed a clear correspondence between the diffusion front of the concentrated protein solution and the localization of the labeled protein (Figure 2a) indicating a process of progressive saturation and specific binding at the diffusion front.The sharply defined diffusion front is a notable feature of the system and suggestive of a reaction-diffusion process. [21]We posited that during the first step (assembly), the high-concentration protein proceeds via diffusion into the arrested clay gel, saturating the clay nanoparticles and restructuring the gel into a proteinclay composite network (Figure 2b, see also Figure 2a, middle column).In the second step (loading), the new reacted protein-clay phase, now saturated by protein, facilitates specific protein binding at the first available unreacted region of clay to create (along with some non-specific binding at the gel surface) a sharply defined protein band at the diffusion front (Figure 2b, see also Figure 2a right column).Hydrogels assembled via an RD process, also known as physical hydrogels, develop characteristic structural features that include, in addition to a distinctive diffusion front, anisotropic arrangements, [22] parallel or perpendicular reorientation of the particles with respect to the diffusion front or flux, [23] rearrangement of the porous network [21] and internal redistribution of the particle density. [24]These features can be studied with a range of imaging techniques, such as brightfield (BF), polarized light microscopy (PLM), and transmission and scanning electron microscopy (TEM and SEM), and all were observable in the protein-clay composite.Under BF, while both reacted and unreacted regions remained transparent, a clearly observable interface at the diffusion front was indicative of a phase transition (Figure 2c).Under PLM, when diffusion was limited to a single plane, a strong and radial birefringence was observed in the reacted-clay region (Figure 2d).A slight polarization was also apparent in the unreacted clay region following autoclave sterilization, but this order was not, we confirmed, required for birefringence arising from RD (Figure S3, Supporting Information).Anisotropic ordering was further examined by TEM, under which Laponite nanoparticles present as spindles of ≈25 nm length [25] due to their low mass thickness contrast when viewed parallel to the planar surface (Figure 2e).Analysis of the particle orientation distribution on the visible axial plane revealed, in the reacted region, preferential alignment perpendicular to the direction of diffusion.We also observed, via particle counts per TEM micrograph frame, a slight increase in the average particle density in the reacted region suggesting some translation of particles counter to the direction of the progressing front in service of the protein-clay complex (Figure S4, Supporting Information).Finally, SEM imaging indicated changes in the microstructure where the reacted region presents as a porous network of fibers and the unreacted remains as a dense granular structure comparable to the pristine clay-gel (Figure 2f; Figure S5, Supporting Information).
These structural features and the process by which they form, indicate a binary RD system.Broadly, a binary RD system is one in which diffusive molecules and a static phase that are initially separated in space, react and develop a diffusion front, the propagation of which leaves behind a reaction product. [26]Several synthetic and naturally occurring macromolecules can self-assemble via binary RD.These types of assemblies typically require a semi-rigid or rigid charged macromolecule [27] to form a static phase (examples include: poly-2,2´-disulfonyl-4,4´-benzidine terephthalamide (PBDT), [21] alginate, [22] -Carrageenan, [28] carboxymethylcellulose, [29] DNA, fibril proteins, [30] chitosan, [31] and curdlan [32] ) which then selfassemble in response to the diffusion of multivalent ions. [7]To our knowledge, this is the first demonstration of an RD assembly system involving an arrested inorganic phase and-perhaps more significantly-where large macromolecules rather than ions constitute the diffusive reactant.It is this feature in particular, facilitated by the very high absorptive capacity of clay, [19] that provides the opportunity to achieve 3D patterning of the loaded protein.
Since the nanoclay-protein gel assembly is the result of an RD process, we expected that characterization of the key parameters known to affect diffusion (concentration, temperature, and time) and clay-protein interactions (such as ionic strength and pH), would enable control of the diffusion front and resultant protein patterning.Correspondingly, the RD rate-as indicated by a progressing diffusion front over time-shows direct dependency on the protein concentration of the assembly solution which continues over time until either complete saturation or upon removal from the assembly solution at which point the RD front is arrested (Figure 3a,c).Variations of the incubation time and protein concentration confirm a linear relationship between the protein diffusion depth and the square root of time, independent of protein concentration (Figure S6, Supporting Information).As expected, assembly at 37 °C accelerates the displacement of the diffusion front compared to 4 °C (Figure S7, Supporting Information).
We found the ionic strength of the assembly solution to be particularly important for the stability of the reacted gel.Notably, BSA alone in salt-free dH 2 0 is, in fact, sufficient to initiate an RD front and sustain gelation, an observation that provides further confirmation that a nanoclay-protein complex forms a reaction product.However, an RD front is observed only at a relatively high protein concentration (≈20 mg mL −1 ).Below this threshold, the rate of swelling of the arrested nanoclay exceeds the rate of RD and the gel dissipates prior to the formation of the clay protein assembly (Figure 3b).The inclusion of ions, either prior to or concurrent with the addition of protein, stabilizes the gel network so that an RD front can be generated at lower protein concentrations (<5 mg mL −1 ) (Figure 3a; Figure S8, Supporting Information).Furthermore, whereas in the presence of salt, the linear dimensions of the gel remain very stable over the entire course of the RD process, in BSA alone the progress of the RD front correlates with a progressive shrinkage of the gel dimensions (Figure S9, Supporting Information).This indicates a further stabilizing influence of ions against protein-driven contraction of the gel network.Finally, increasing ionic concentration exerted a slight influence on the RD rate where an increase in salt concentration up to ≈50 mm correlated with faster progression of the RD front (Figure S10, Supporting Information).
It is notable that both the ionic and protein concentrations applied in these experiments go well above the threshold at which Laponite suspensions characteristically undergo flocculationinduced phase separation in simple mixed systems. [33]Such flocculation is caused by cation-induced compression of the negative electric double layer on the particle surface and a resultant accumulation of face-to-edge attractive interactions between particles.We have previously proposed a diffusion gelation mechanism in which a progressively increasing solute concentration across an arrested nanoclay phase causes local reorientation and electrostatic bonding of particles into a stable gel network, resistant to further translation of particles into large-scale flocculates. [34]The current results are broadly consistent with this conceptualization but indicate discrete contributions of ions and proteins.While ion diffusion plays a role in stabilizing the network, the formation of the sharply defined diffusion front indicates that threshold concentrations of diffusing proteins effect a further phase transition in the gel structure.Further experimental and theoretical studies are warranted to characterize these interactions and the anisotropic structures that result.
The influence of pH on gel assembly appears more subtle and is likely to be protein-specific.In the case of BSA, like ionic concentration, increasing pH between 4 and 10 correlated with a slight but significant increase in the rate of progression of the RD front (Figure S10, Supporting Information), but unlike ionic concentration did not cause a significant change in the gel dimensions over an equivalent time frame (p = 0.516).Both ionic strength and pH effects on protein-clay interactions are complex and change both system components in a variety of non-linear ways.The increase in the rate of front progression with pH and salt concentration would be consistent with a reduction in the saturation capacity of clay surfaces due to steric effects arising from protein conformational changes or else altered electrostatic profiles. [35,36]us, pre-treatment of a Laponite colloid with high concentration BSA protein in a salt buffer causes a change in the state of the Laponite (to the reacted state) which then allows patterning of a subsequently added protein at the reaction front.To determine if the induction of the reacted state was specific to BSA or could be extrapolated across a wider range of proteins, we next performed assembly with various globular proteins across a spectrum of isoelectric points (4-10) and molecular weights (14-150 kDa) before loading with FITC-BSA.In every case a distinct diffusion front was generated, although the RD rate varied significantly between proteins, along with the opacity of the reacted gel (Figure S11, Supporting Information).Interestingly, the rate correlated poorly with either protein size or net charge, and further work is therefore required to understand the specific parameters influencing each protein's RD rate in this system, as well as the effect of different proteins on the reacted gel structure.
The ability to load alternative proteins at the reaction front once assembled, appears to be dependent principally on the charge of the loaded protein in relation to that of the assembling protein (Figure 4a).So, for example, in gels assembled in lysozyme, loaded lysozyme (isoelectric point, 11.35) localized at the diffusion front whereas in gels assembled in BSA (isoelectric point, 4.7), lysozyme binding was limited to the gel surface-a result also observed when BSA was loaded into lysozyme assembled gels (Figure 4a).These results imply a selective diffusion process governed by electrostatic interactions between the proteinclay complex and the loaded proteins in which attractive interactions hinder diffusion and repulsive interactions facilitate it.Correspondingly, we found that lysozyme loading at the reaction front could be achieved even in a BSA-assembled gel by lowering the pH of the assembly buffer to the point where both BSA and lysozyme were positively charged (Figure 4b).A related effect of pH on loading was the observation that the "nonspecific" binding of FITC BSA at the gel surface (as opposed to at the diffusion front; cf. Figure 1c) could be eliminated by lowering the pH of the assembly solution (Figure S10, Supporting Information).This non-specific surface binding can be accounted for by the altered charge-related surface properties of BSA when labeled with FITC (such as a lower isoelectric point (IEP) and increased hydrophobicity relative to native BSA).These changes are well characterized in the literature [37] and are sufficient to explain the secondary binding of a fraction of the loaded labeled protein within regions already reacted with unlabeled BSA.Attenuating these effects of FITC labeling by lowering the pH toward the protein IEP thus also attenuates secondary binding.
As noted, the use of proteins as diffusive reactants presents an exciting opportunity for protein patterning through scalable, bottom-up assembly methods.By effecting a controllable phase transition in the colloidal gel structure, proteins can be stably "written-in" by the progressing RD front allowing a unique degree of control and stability not achievable with other diffusionbased hydrogel functionalization strategies. [9,10]Thus, by timed introduction into the assembly solution, loaded proteins can be patterned into the assembled gel at both very high concentrations (in the order of tens of milligrams per mL (Figure S13, Supporting Information)) and at very high resolution.It was relatively simple, for example, by lowering the assembly protein concentration to slow the RD rate to achieve protein band The localization of proteins at the diffusion front is driven by the charge of the loaded protein with respect to the charge of the protein used for nanoclay-protein gel assembly.BSA and lysozyme (10 mg mL −1 ) in PBS (pH 7.4) possess negative and positive net charges, respectively.When using the same type of protein for assembly and loading, the loaded protein (100 μg mL −1 FITC BSA or FITC lysozyme) localized at the diffusion front, and when using oppositely charged proteins they localized at the gel surface.b) The protein charge of the assembly solution can be altered by adjusting the pH buffer to facilitate its diffusion to the diffusion front so in contrast to clay-gels assembled with BSA pH 7 which resulted in lysozyme binding only at the surface, gels assembled with BSA at pH 3 before exposure to FITC-lysozyme allowed lysozyme loading at the diffusion front.c) Proteins are stably bound within the reacted region allowing the creation of sequential protein bands by alternating the assembly and loading steps.Here scaffolds were loaded with repeated bands of FITC-BSA or with alternating bands of BSA (labeled with Alexa-633) and lysozyme (labeled with Alexa-488).d) Similarly, by changing the protein concentration of diffusive protein as a function of time both positive and negative gradients of varying magnitude could be simultaneously generated.e,f) Varying the assembly time allows a high degree of control over the localization of loaded protein distribution, down to f, sub-cellular resolutions of band spacing (1.99 ± 0.37 μm) and thickness  thickness and spacing at sub-cellular resolutions approaching that of two-photon patterning, the current state of the art [38] (Figure 4e,f).
Unusually for a physical hydrogel, we found that once incorporated at the RD front, loaded proteins can remain localized for extended periods in ionic solution without being displaced even under agitation (Figures S2 and S12, Supporting Information).This high degree of stability, while not fully understood, is consistent with the high sorption capacity of clays for proteins as well as their well-established ability to stabilize biomolecules through surface binding-phenomena of known significance in soil ecosystems. [39] controlling the order of introduction, timing, and concentration of loaded protein with respect to the assembly process, it was possible to generate a range of conformations of protein patterning.So, for example, gels of multiple, punctuated bands of loaded protein could be achieved by repeatedly alternating high protein concentration assembly steps with lower concentration loading steps.With careful timing and buffer selection, alternating bands of different proteins could also be achieved using this approach (Figure 4c).Similarly, by gradually changing the concentration of loaded protein within the assembly solution over the course of the RD process, continuous gradients of varying magnitude (depending on the rate of change) and dual gradients of simultaneously increasing and decreasing protein concentrations, could be patterned with high reproducibility within the gel structure (Figure 4d).
Finally, we sought to test the ability of a protein patterned through the RD system to induce a biological response.Our previous work [20] has demonstrated the utility of nanoclay gels for sustaining and localizing the activity of the bone-inducing morphogen, bone morphogenic protein 2 (BMP-2) in vivo and so here the ability of RD patterned BMP-2 to template bone formation was tested.BMP-2 was labeled with Dylight 633 to allow visualization of patterning within the gel and activity was confirmed using the C2C12 dose-response assay (Figure S14, Supporting Information).A serendipitous finding of this study was that labeling with the negatively charged Dylight molecule was itself sufficient to allow patterning, even at neutral pH, of the otherwise positively charged BMP-2 protein (pI = 8.5) within a BSA assembled gel (Figure S15, Supporting Information).BSA RD gels were patterned with a sharply defined band of BMP-2 spatially distinct from the gel surface (Figure 5b).We would stress that the purpose of this design was specifically to test the hypothesis that resultant bone induction would conform to the geometry of the patterned BMP-2 and was not intended as a biological optimum for BMP-2 delivery.Patterned BMP-2 gels were assessed for bone induction in a murine subcutaneous ectopic bone induction assay against gels in which an equivalent concentration of BMP-2 + BSA was mixed homogenously throughout the gel volume.μCT and histological analysis revealed bone formation in 4/5 patterned samples but only 2/5 mixed gels within the 8week time frame (Figure S16, Supporting Information).As well as differences in BMP2 distributions that yield ≈6× higher local concentration within patterned regions of the RD gels, we also noted that the mixed preparations displayed a lower elastic modulus that may also be a relevant difference affecting bone forming efficiency (Figure S17, Supporting Information).No bone was present in the absence of BMP-2.
Histological analysis confirmed new bone spicule formation proximal to the implanted gel volume (Figure 5d; Figure S17, Supporting Information).Critically, region of interest analysis of bone formation in 8-week explants confirmed >90% of bone in the patterned gels localized to within 0.4 mm of the expected BMP-2 region (compared with <5% in the mixed gels) (Figure 5e).Furthermore, where sufficient bone tissue permitted measurement of curvature (n = 3), the geometry of the bone tissue conformed tightly to that of the labeled protein imaged prior to implantation (Figure 5g).These results thus provide strong evidence for the ability of RD gels to pattern active BMP-2 to guide bone formation in vivo.

Conclusion
In summary, we have reported a new binary reaction-diffusion system where, uniquely, macromolecules rather than multivalent ions function as the diffusive reactant to restructure an arrested inorganic colloid into a stable gel.Employing this phenomenon, we show how, through timed introduction over the course of the reaction-diffusion process, functional proteins can be stably patterned into the resultant gel structure and demonstrate the utility of this approach for patterning the morphogen BMP-2 to template bone induction in vivo.The approach has sig-nificant advantages.As a bottom-up self-assembly method, patterning is highly scalable and can be achieved under physiological and biocompatible conditions, free of chemical cross-linkers, and employing only (if necessary) clay interactions with proteins and ions present in the blood.Active proteins can be patterned at resolutions comparable to the limits of current fabrication techniques, at concentrations in the order of tens of milligrams per mL, and with patterns remaining stable in solution over several years.These advantages, combined with the established ability of nanoclays to enhance growth factor bioactivity in vivo, suggest exciting potential for high-resolution control of biological function at clinically relevant scale.

Experimental Section
All experiment details are provided in the Supporting Information.

Figure 1 .
Figure1.Assembly of 3D protein patterning within nanoclay colloidal gels.a) Laponite clay nanoparticles approximate 25 nm disks of 1 nm thickness and possess a permanent negative surface charge and a weak positive edge charge at neutral pH that forms viscous colloidal dispersions in water above ≈2 wt.%.b) After drop-casting a viscous dispersion of Laponite into a high-concentration protein solution (concentrations in the order of 1-100 mg mL −1 ) and then, after a given time, transferring the gel to a lower concentration solution (concentrations in the order of 1-100 μg mL −1 ) of labeled protein a sharply defined band of labeled protein within the gel was observed.c) For example, 3D reconstruction of confocal image and analysis of transparent gel droplets exposed to 10 mg mL −1 BSA for 1 h and then 100 μg mL −1 FITC BSA for 1 h reveals a sharp band of fluorescent protein patterned between the gel periphery and core (scale bar, 200 μm), the plot presents fluorescent intensity within a linear region of interest (running from top left to the bottom right of the image.d) The system supports the assembly and patterning of structures of a range of size and shapes as defined by the initial casting.For example, UV transilluminator images of i) clay-gel droplets of 20 μL incubated in 60% FCS PBS for 170 min and then in 100 μg mL −1 FITC BSA for 48 h (scale bar, 200 μm); ii) 1 mL clay-gel star-shape assembled in 50 mg mL −1 BSA PBS for 4 h and patterned with 500 μg mL −1 FITC BSA for 24 h; iii) a segmented 1 mL clay-gel cylinder assembled in 100% FCS for 72 h and patterned with 250 μg mL −1 FITC BSA for 96 h (scale bars, 4 mm).Similar results were obtained for n = 3 independent samples.

Figure 2 .
Figure 2. Reaction-diffusion phenomena in nanoclay-protein assembly and patterning.a) Confocal analysis of nanoclay colloidal gels loaded with FITC-BSA without, concurrent with, or after assembly in BSA (10 mg mL −1 ).Inclusion of FITC BSA in the assembly solution reveals a protein gradient (yellow arrow) with a clear diffusion front (red arrow) that co-locates with the fluorescent band formed when FITC-BSA is loaded after assembly in BSA (n = 3, scale bars, 200 μm).b) A Schematic illustration of the proposed nanoclay-protein assembly process in which high-concentration protein progressively saturates the clay nanoparticles to facilitate specific binding of the loaded protein at the diffusion front.c-f) Microscopy analysis confirms a phase transition occurs behind the diffusion front.c) Bright-field images of gels reveal the formation of an interface at the diffusion front indicative of a phase transition (n = 3, scale bars, 200 μm).d) Polarized light microscopy reveals radial birefringence of the reacted clay region as well as threaded texture in the unreacted clay (n = 3, scale bars, 200 μm).e) Transmission electron microscopy indicates a preferential orientation of the nanoparticles behind the diffusion front in contrast to a random orientation in the non-reacted region (n = 3, scale bars, 100 nm).f) SEM shows a more open, porous structure in the reacted region compared to the unreacted core (n = 3, scale bars, 1 μm).

Figure 3 .
Figure 3. Nanoclay-protein gel reaction-diffusion rate as a function of protein concentration and the presence of ions.a) Confocal micrographs reveal a first fluorescent band at the surface of all nanoclay-protein gel and a second fluorescent band that displaces toward the core with an increase in the protein concentration indicating a more rapid progression of the diffusion front.b) A diffusion front was also generated without salts, but only at protein concentrations of 20 mg mL −1 or above as below this the arrested nanoclay dispersion swelled and dissipated prior to the formation of a clay-protein assembly.c,d) A linear regression indicates that the displacement of the internal fluorescent ring with and without ions is concentration-dependent with an R 2 of 0.99 and 0.98, respectively.The results represent mean ± SD for n = 4 and the scale bars to 200 μm.

Figure 4 .
Figure 4. Versatility of nanoclay-protein gel patterning.a)The localization of proteins at the diffusion front is driven by the charge of the loaded protein with respect to the charge of the protein used for nanoclay-protein gel assembly.BSA and lysozyme (10 mg mL −1 ) in PBS (pH 7.4) possess negative and positive net charges, respectively.When using the same type of protein for assembly and loading, the loaded protein (100 μg mL −1 FITC BSA or FITC lysozyme) localized at the diffusion front, and when using oppositely charged proteins they localized at the gel surface.b) The protein charge of the assembly solution can be altered by adjusting the pH buffer to facilitate its diffusion to the diffusion front so in contrast to clay-gels assembled with BSA pH 7 which resulted in lysozyme binding only at the surface, gels assembled with BSA at pH 3 before exposure to FITC-lysozyme allowed lysozyme loading at the diffusion front.c) Proteins are stably bound within the reacted region allowing the creation of sequential protein bands by alternating the assembly and loading steps.Here scaffolds were loaded with repeated bands of FITC-BSA or with alternating bands of BSA (labeled with Alexa-633) and lysozyme (labeled with Alexa-488).d) Similarly, by changing the protein concentration of diffusive protein as a function of time both positive and negative gradients of varying magnitude could be simultaneously generated.e,f) Varying the assembly time allows a high degree of control over the localization of loaded protein distribution, down to f, sub-cellular resolutions of band spacing (1.99 ± 0.37 μm) and thickness (4.6 ± 0.25 μm).Images are representative of n = 3 and the scale bars a-d) 200 μm; e) 50 μm, and f) 10 μm).
Figure 4. Versatility of nanoclay-protein gel patterning.a)The localization of proteins at the diffusion front is driven by the charge of the loaded protein with respect to the charge of the protein used for nanoclay-protein gel assembly.BSA and lysozyme (10 mg mL −1 ) in PBS (pH 7.4) possess negative and positive net charges, respectively.When using the same type of protein for assembly and loading, the loaded protein (100 μg mL −1 FITC BSA or FITC lysozyme) localized at the diffusion front, and when using oppositely charged proteins they localized at the gel surface.b) The protein charge of the assembly solution can be altered by adjusting the pH buffer to facilitate its diffusion to the diffusion front so in contrast to clay-gels assembled with BSA pH 7 which resulted in lysozyme binding only at the surface, gels assembled with BSA at pH 3 before exposure to FITC-lysozyme allowed lysozyme loading at the diffusion front.c) Proteins are stably bound within the reacted region allowing the creation of sequential protein bands by alternating the assembly and loading steps.Here scaffolds were loaded with repeated bands of FITC-BSA or with alternating bands of BSA (labeled with Alexa-633) and lysozyme (labeled with Alexa-488).d) Similarly, by changing the protein concentration of diffusive protein as a function of time both positive and negative gradients of varying magnitude could be simultaneously generated.e,f) Varying the assembly time allows a high degree of control over the localization of loaded protein distribution, down to f, sub-cellular resolutions of band spacing (1.99 ± 0.37 μm) and thickness (4.6 ± 0.25 μm).Images are representative of n = 3 and the scale bars a-d) 200 μm; e) 50 μm, and f) 10 μm).

Figure 5 .
Figure 5. Reaction-diffusion patterning of BMP-2 templates ectopic bone induction.a) Nanoclay gels (50 μL) were assembled in BSA to pattern BMP-2 before murine subcutaneous implantation within a space retaining polylactic acid scaffold.RD patterned gels contained 9.95 ± 0.42 μg mL −1 BMP-2 (Dylight NHS 633, ELISA, n = 2) and 15.89 ± 3.28 mg mL −1 BSA (protein assay, n = 3) and were compared with controls containing equivalent protein concentrations of either BMP-2 and BSA or BSA alone through mixing.b) Confocal micrographs and respective ROI show the spatial localization of labeled BMP-2 within the nanoclay-gels.RD patterned BMP2 was localized as a band 0.92 ± 0.09 mm from the gel surface (n = 3, scale bar 200 μm).c) The average structure diameter, length, and height of the assembled was highly uniform (4.68 ± 0.04, 4.64 ± 0.03, and 2.51 ± 0.18 mm (n = 6), scale bar 1 mm).d) Robust formation of bone within the gel cylinder region in the patterned gels was apparent by week 4 post implantation (scale bar 1 mm).Alcian blue/Sirius red (A/S) staining confirms cell invasion and a distinct band of ectopic bone formation within the patterned gel (scale bar 200 μm).e) Micro CT analysis of specific bone within the expected ROI (⌀ 2.8 mm ± 20%) reveals more bone formation over the projected area of the BMP-2 pattern compared to the positive control (n = 3, scale bar 1 mm).f) Measurement of the bone curvature corresponds closely with that of the BMP-2 pattern observed prior to implantation as confirmed through CT analysis (n = 3, scale bar 1 mm) and illustrated against Sirius red staining of new bone (scale bar 200 μm).