A bio-inspired swellable microneedle adhesive for mechanical interlocking with tissue

Abstract

Achieving significant adhesion to soft tissues while minimizing tissue damage poses a considerable clinical challenge. Chemical-based adhesives require tissue-specific reactive chemistry, typically inducing a significant inflammatory response. Staples are fraught with limitations including high-localized tissue stress and increased risk of infection, and nerve and blood vessel damage. Here inspired by the endoparasite Pomphorhynchus laevis, which swells its proboscis to attach to its host's intestinal wall, we have developed a biphasic microneedle array that mechanically interlocks with tissue through swellable microneedle tips, achieving ~3.5-fold increase in adhesion strength compared with staples in skin graft fixation, and removal force of ~4.5 N cm^-2 from intestinal mucosal tissue. Comprising a poly(styrene)-block-poly(acrylic acid) swellable tip and non-swellable polystyrene core, conical microneedles penetrate tissue with minimal insertion force and depth, yet high adhesion strength in their swollen state. Uniquely, this design provides universal soft tissue adhesion with minimal damage, less traumatic removal, reduced risk of infection and delivery of bioactive therapeutics.

ARTICLE

Received 27 Nov 2012 | Accepted 6 Mar 2013 | Published 16 Apr 2013

Seung Yun Yang1,2,3, Eoin D. OCearbhaill1,2,3, Geoffroy C. Sisk4, Kyeng Min Park5, Woo Kyung Cho1,3, Martin Villiger6, Brett E. Bouma3,6, Bohdan Pomahac4 & Jeffrey M. Karp1,2,3

Achieving signicant adhesion to soft tissues while minimizing tissue damage poses a considerable clinical challenge. Chemical-based adhesives require tissue-specic reactive chemistry, typically inducing a signicant inammatory response. Staples are fraught with limitations including high-localized tissue stress and increased risk of infection, and nerve and blood vessel damage. Here inspired by the endoparasite Pomphorhynchus laevis, which swells its proboscis to attach to its hosts intestinal wall, we have developed a biphasic microneedle array that mechanically interlocks with tissue through swellable microneedle tips, achieving

B3.5-fold increase in adhesion strength compared with staples in skin graft xation, and removal force of B4.5 N cm 2 from intestinal mucosal tissue. Comprising a poly(styrene)-block-poly(acrylic acid) swellable tip and non-swellable polystyrene core, conical micro-needles penetrate tissue with minimal insertion force and depth, yet high adhesion strength in their swollen state. Uniquely, this design provides universal soft tissue adhesion with minimal damage, less traumatic removal, reduced risk of infection and delivery of bioactive therapeutics.

1 Division of Biomedical Engineering, Department of Medicine, Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

2 Harvard Stem Cell Institute, 1350 Massachusetts Avenue, Cambridge, Massachusetts 02138, USA. 3 Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. 4 Department of Surgery, Division of Plastic Surgery, Brigham and Womens Hospital, 75 Francis Street, Boston, Massachusetts 02115, USA. 5 Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA. 6 Wellman Center for Photomedicine, Harvard Medical School and Massachusetts General Hospital, 40 Blossom Street, Boston, Massachusetts 02114, USA. Correspondence and requests for materials should be addressed to J.M.K. (e-mail: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms2715

A bio-inspired swellable microneedle adhesive for mechanical interlocking with tissue

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2715

Achieving signicant levels of adhesion to soft tissues presents considerable challenges, especially when tissues are wet1,2. Chemical adhesives, such as cyanoacrylate,

adhere strongly to tissues by a reactive exothermic covalent crosslinking reaction. However, these adhesives often cure too fast or too slowly and release toxic degradation products such as formaldehyde, leading to an intense inammatory response3. While clinically utilized brin glue can effectively bond to wet tissue without a signicant inammatory response, it exhibits low strength of adhesion due to poor cohesive properties4, and can be challenging to apply as the polymerization time is difcult to control during placement. Alternatively, biocompatible hydrogel adhesives5,6 that covalently bond to specic tissues can achieve a signicant level of adhesion, yet their effectiveness depends on the presence of surface biomolecules with specic functional groups (e.g., NH2, SH or COOH). Therefore, the chemistry must be tailored for each tissue, and hydrogel adhesives do not adhere strongly to tissues such as skin. Also, it is critical to note that adhesives that rely on chemical bonding can easily be fouled in the presence of blood, thus compromising their efcacy in many surgical settings. For universal use, gold standard sutures and staples based on mechanical xation are widely used79, however their placement can extend operating times as the tissue often must be manipulated before each pass of a suture needle. Sutures are difcult to place in small spaces (that is, during laparoscopic procedures)10, and do not work effectively for repair of many tissues including the dura mater7, urethral defects8 and lung tissue9. While staples are typically quicker and easier to place than sutures, they can cause signicant tissue damage, scarring, and excessive depth of tissue penetration can result in nerve and blood vessel damage11. Staples also pose a higher risk of developing a wound infection compared with sutures12. There are several unmet clinical needs for adhesives to afx connective tissues, including tendons and ligaments, to improve contact between tissues to reduce motion of tissue grafts, and to seal tissues for prevention of uid (intestine) or air (lung) leaks. New adhesives are also needed to prevent formation of seromas following operations that create dead space between layers of tissue (for example, abdominoplasty), thus avoiding the need for drains that increase the risk of infection and potential necessity for reoperation13.

A suitable platform approach to overcome the limitations of existing adhesives should ideally avoid the use of reactive chemistry, provide strong tissue adhesion in both normal and shear directions, be amenable to quick application, be simple to position over the target site, be removable (or degradable) with minimal tissue damage, provide adhesion to dynamic tissue surfaces while withstanding multiple extension/compression cycles, minimize the risk of infection and have the capacity to deliver therapeutics. Compared with glues, sutures and staples, tissue adhesive tapes have shown signicant advantages including reduced procedure times, reduced scarring, ability to spread tissue forces over a larger surface area and enhanced tissue handling1416. Additionally, adhesive tapes may be useful as an internal equivalent of a transdermal drug delivery patch to deliver drugs such as antibacterial, proregenerative or anti-inammatory agents to target tissues. While barrier membranes and hernia meshes are routinely used clinically, these typically provide minimal adhesion, and often several sutures or tacks are used for xation. Tape adhesives with hook-like protrusions have been developed, however their adhesion to tissue is fundamentally limited to surface entanglement without penetration of the tissue17.

In seeking an optimal method of universal soft tissue adhesion with minimal tissue damage, we looked for inspiration from living organisms that have through the course of evolution

adapted this function. Endoparasitic worms known as spiny-headed worms use a proboscis to penetrate through tissue. Species, such as Pomphorhynchus laevis, secure rm anchorage to the sh intestinal wall by expanding a bulb using retractor muscles at the base of the proboscis following penetration18. Using the adaptable morphology of the worm proboscis, we looked to create a structured biphasic microneedle (MN) with optimal characteristics for needle insertion and retention in tissue.

Here we demonstrate a highly engineered MN with a shape change swellable tip that facilitates mechanical interlocking with tissue (Fig. 1a). This design minimizes the force needed for tissue penetration, as the smooth, cone-shaped needles can be inserted into tissue in a dry (stiff) state and do not include protruding barb features, associated with other proposed MN-based adhesive platforms19. Signicant pull-out force can be achieved through the rapid increased cross-sectional area that occurs with swelling at the needle tips on contact with water (in tissue), leading to localized tissue deformation and subsequent interlocking. The bio-inspired MN adhesive showed high levels of adhesion with wet tissues, such as skin and intestine tissue, regardless of differences in surface texture. Additionally, strong xation can be achieved in dynamic tissues during multiple cycles of movement and as increased pull-out forces are achieved via swelling within tissue, the soft MN tips (that is, reduced modulus) enable removal without signicantly damaging the tissue. Furthermore, unlike stiff MNs that may break during removal from tissue, the modulus change due to swelling prevents breakage of the swollen MNs.

ResultsBio-inspired MN array. To create a stimulus responsive MN platform with low penetration force and strong adhesion, we pursued a double-layered MN design with selective localization of swellable material in the tip region. Following preferential distal swelling, each MN had both soft (outer) and stiff (inner) regions. In addition to having a MN platform comprising both swellable and non-swellable components, we sought to control the mechanical and water permeation properties of the swellable material, while promoting signicant interaction between the soft outer layer and the stiff inner layer to prevent delamination. For this purpose, we considered an amphiphilic block copolymer (BCP) design that exhibits selective responsiveness to stimuli such as the presence of aqueous or organic solvents20,21. We envisioned that dual hydrophilichydrophobic properties of the outer layer would enable rapid absorption of water and promote intimate interaction with the non-swellable inner hydrophobic region. Block copolymers contain two or more chemically different polymers connected by covalent bonds that offer a means of combining the desirable characteristics of different polymers into a hybrid material22. The mechanical properties and swellability of the outer region of the MNs can be controlled by manipulating the overall average molecular weight of the polymer and the weight fraction of each block20,23. In this study, we chose a polystyrene-block-poly(acrylic acid) (PS-b-PAA) block copolymer as the swellable material and PS homopolymer as a non-swellable material. PAA is a well-known super-absorbent polymer used in several biomaterials-based strategies and in consumer products such as diapers that possesses COOH groups that quickly become ionized in the presence of water. In contrast, PS exhibits mechanical strength and structural integrity without swelling. As predicted, high volume expansion occurred when PAA was the major block component (over 70% in weight fraction). The mechanical robustness of the block copolymer could be strengthened by increasing the weight fraction of the PS block

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2715 ARTICLE

a

b c

PS-b-PAA

PS

Non-swellable

PS-b-PAA

casting

Swellable

PDMS mould

PS-b-PAA

Epidermis

Dermis

Mechanical interlocking

n

PS filling

under vacuum

H2O

26 kg mol1 76 kg mol1

(wtPS 25%)

O

HO

+ H2O

Peeling off

from mould

m

d e

h

f g

Figure 1 | Concept and fabrication of the bio-inspired microneedle (MN) adhesive. (a) Illustration showing mechanical interlocking of a water-responsive shape-changeable microneedle following penetration into a tissue. (b) Schematic showing the preparation of the double-layered MN array using a PDMS mould and showing the chemical structure of PS-b-PAA with PS weight fraction (wtPS) of 25% used for the swellable tip. (c) Cartoon showing the inner structure of double-layered MN and reversible water responsiveness. A thin hydrophobic lm comprising the PS block covered the outer surface of the MN, likely due to the presence of the hydrophobic PDMS mould. During lling of the non-swellable PS core, PS chains likely entangle with the PS block at the interface (dashed line) between the swellable tip and PS core, providing interfacial adhesion to prevent delamination. (df) Side view images of hollow MN (without the PS core) with different PS-b-PAA lling fractions, including (d) 20%, (e) 40% and (f) 70% (height of PS-b-PAA layer compared with total MN height). These MNs were prepared by solvent-casting PS-b-PAA dissolved in DMF using different concentrations (d: 10 wt%, e: 18 wt% and f: 25 wt%). (g) Double-layered MN with a swellable tip (20% height fraction) following lling of the PS core. Scale bar, 200 mm. (h) Photograph of the double-layered

MN array with density of 10 10 cm 2 showing high-pattern delity. Scale bar, 1 mm. The PS-b-PAA tip is clearly distinguishable from the PS core as

shown in the inset (Scale bar, 500 mm).

(Supplementary Fig. S1). Interestingly, the stiffness of the block copolymer in its swollen state, with PS of B25% weight fraction, approximates that of skin or intestine tissue (Supplementary Fig. S1), which reduces the risk of underlying tissue damage without signicantly compromising the interlocking mechanism. To permit rapid water penetration into the PS-b-PAA layer when the PS block preferentially assembles at the air-copolymer surface, we selected a PS block with a relatively low-molecular weight (o100,000 g mol 1)

that minimizes interfacial surface area coverage due to short polymer chains. To achieve this, PS-b-PAA was prepared from the hydrolysis of polystyrene-block-poly(tert-butyl acrylate) (PS-b-PtBA) with number-average-molecular weights (Mn) of 26,000 g mol 1 for PS and 128,000 g mol 1 for PtBA. Complete conversion of PtBA to

PAA was conrmed by nuclear magnetic resonance (NMR) (Supplementary Fig. S2). For functional testing in tissue, we selected swellable tips fabricated with PS-b-PAA with Mn of

26,000 g mol 1 and 76,000 g mol 1 for PS and PAA, respectively, and a PS weight fraction of B25%.

As shown in Fig. 1a,b a double-layered MN array having water-swellable tips was fabricated by solvent-casting a PS-b-PAA solution in N, N-Dimethylformamide (DMF) on a PDMS (polydimethy lsiloxane) mould with a 10 10 array of conical cavities within a

1 cm2 area. Drying of the solvent prompted the formation of a thick lm at the tip region of the conical cavity via capillary forces and a thin lm on the remainder of the mould. To form the MN inner core and backing layer of the lm, the PS homopolymer was melted on top of the continuous PS-b-PAA layer at 180 C under vacuum. After cooling to room temperature, the double-layered conical MN array was carefully peeled from the PDMS mould.

Macroscopically, the double-layered BCP MN exhibits a needle-in-needle structure consisting of swellable outer PS-b-PAA layer and supporting PS inner core (Fig. 1c). Microscopically, the PS-b-PAA layer self-assembles into multiple phases whose location and properties are dependent on the interaction with the mould and air surface. To examine the composition of the swellable polymer surface at the air and PS core interfaces, we performed X-ray photoelectron spectroscopy (XPS) on PS-b-PAA lms that were casted onto PS and PDMS substrates (Supplementary Fig. S3). Given that the theoretical C/O ratio for PAA is 1.5, the C/O ratio at the surface of the PS-b-PAA layer on PDMS (14.9) and PS (22.3) suggests that PS was the dominant interfacial block. Thus, we speculate that a loosely packed PS lm with a thickness of B10 nm forms at the surface of the PDMS mould (air interface following removal from the mould) and modulates the rate of diffusion,

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2715

a

b

0 s

60 s

600 s

Insertion

0 s

Gel

In tissue

120 s

360 s

600 s

Figure 2 | Swelling of BCP MN following insertion into a hydrogel and muscle tissue. (a) Time-dependent swelling of the BCP MNs (40% swellable tip height fraction) following insertion into a 1.4 wt% agarose hydrogel (0, 60 and 600 s). (b) OFDI images showing swelling of the same BCP MNs following insertion into muscle tissue (0, 120, 360 and 600 s). Scale bar, 500 mm.

preventing a rapid modulus drop caused by immediate water absorption, which would result in insertion failure of the BCP MN array. Furthermore, during lling of inner core by melted PS, the PS block in the PS-b-PAA layer can entangle with a PS homopolymer core and the COOH groups in the PAA chains thermally crosslink via intermolecular anhydride formation24,25. By varying the concentration of the casting solution, the height of the PS-b-PAA layer was controlled to be either 20, 40 or 70% of BCP MN height (Fig. 1df). Importantly, the PS base material completely lled the inner cores showing good contact with the PS-b-PAA layer (Fig. 1g). Figure 1h shows macroscale images of a double-layered BCP MN array with high-pattern delity having a density of 10 10 cm 2, and having a swellable tip height

of 20%, a MN base diameter of 280 mm and a MN height of 700 mm. An overall needle height of 700 mm was selected to ensure that the needle tip swelling would occur beneath the surface of the tissue (for example, thickness of epidermis is less than 100 mm at typical needle insertion sites)26 and to minimize the penetration depth.

Reversible shape change of a water-responsive swellable MN. To visualize the real-time shape change of swellable BCP MN, we monitored the swelling behaviour immediately following insertion into a transparent agarose hydrogel (see Supplementary Fig. S4 for experimental setup). Once BCP MNs were inserted into the hydrogel (tip height B40% of total MN height), the volume of the PS-b-PAA tip layer rapidly increased and reached

B60% of the maximum swollen state within 1 min. Subsequently, swelling equilibrated to a maximum swollen state within 10 min (Fig. 2a and Supplementary Movie 1). The kinetics of water absorption of micron-sized PS-b-PAA were much faster than

those of bulk material with mm-scale thickness (Supplementary Fig. S1), which was likely the result of the high surface area per volume ratio for the MN tips and short water diffusion length. At equilibrium, the volume of PS-b-PAA MN layer had expanded to B9 times its initial volume (Supplementary Fig. S5). Volume expansion of BCP MN by swelling predominantly occurred in the tip region and swollen BCP MN interlocked with the hydrogel. The interlocked BCP MN could be removed from agarose gel without breakage or delamination. The swollen tips immediately began to deswell following retrieval from the hydrogel substrate and fully recovered to their original conical structure within 15 min (Supplementary Fig. S6 and Supplementary Movie 2).

When PS or PAA homopolymers were used as the MN tip material, they produced opposing results. The non-swellable PS MN was easily inserted into the hydrogel and freely removed without signicant applied force, whereas the PAA MN did not penetrate the hydrogel because of low mechanical strength (Supplementary Fig. S7). Although it is possible to make stiff PAA MN that can be inserted into hydrogel (or tissue) by crosslinking following harsh thermal treatment, pure PAA MN is also prone to delamination during removal of the MN due to poor interfacial adhesion with the backing layer.

To verify the stimulus-responsive mechanical interlocking of the BCP MN in animal tissue, we utilized optical frequency domain imaging (OFDI) that is capable of providing non-invasive cross-sectional views of internal tissue structures to a depth of 12 mm with high resolution27. While the BCP MN inserted into muscle tissue showed less initial volume expansion rate compared with the agarose hydrogel, it reached the same nal swollen state within 10 min (Fig. 2b and Supplementary Movie 3). Interestingly, insertion into muscle tissue induced radial expansion of the BCP MN compared with axial swelling within the homogeneous agarose hydrogel. As observed within muscle tissue via OFDI, the swollen BCP MN formed a mushroom-shaped structure that provides a more favourable geometry to support mechanical interlocking with tissue.

Firm adhesion of the swellable MN adhesive. The adhesive properties of the BCP MN were evaluated by testing the normal adhesion strength of a 10 10 MN array (1 cm2 cross-sectional

area) on fresh cadaveric porcine skin. We investigated the effect of swelling-induced MN shape change on adhesion, compared with the adhesion of non-swellable PS MN (Fig. 3a). Adhesion of at PS-b-PAA (BCP) and PS lms were also measured. Both at-surfaced BCP and PS lms showed low adhesion strength to skin (o0.1 N cm 2). While, BCP MN and PS MN showed similar force versus displacement proles during skin insertion (Supplementary Fig. S8) and exhibited similar adhesion strength to skin immediately after insertion, after 10 min the maximum swollen state for BCP MN was achieved and the adhesive strength of the BCP MN dramatically increased. BCP MN with a swellable tip height of 20% (BCP MN (20%)) showed B7 times higher adhesion strength (0.690.17 N cm 2) than PS MN adhesive (0.0980.015 N cm 2). Furthermore, for BCP MN with a swellable tip height of 40% (BCP MN (40%)), the adhesive strength increased B12-fold (1.230.26 N cm 2). However, MN with a swellable tip height of 70% (BCP MN (70%))

exhibited reduced adhesive performance (Supplementary Fig. S9), likely due to a less favourable conguration for interlocking with tissue. No signicant increase in adhesive strength was observed for the BCP MN for longer than 30 min swelling (Fig. 3b). For most intended uses, a short application time is desirable; therefore, the swelling time was standardized at 10 min for all additional experiments.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2715 ARTICLE

a

b c

1.8

1.41.21.00.80.60.40.20.0

2.0 4

2 min

1.6

Adhesion strength (N cm2 )

Adhesion strength (N cm2 )

1.6

0.8

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PS film

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BCP MN (40%)

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25 30 360

2 (iii) (iv)

(i)

(ii)

0

2.0

Insertion

Pull-out

1.5 1.0 0.5 0.0

Position from skin surface (mm)

d e

60

Figure 3 | MN adhesive rmly adheres to skin. (a) Normal adhesion strength for PS MN and BCP MN adhesives with 20 and 40% swellable tip height fractions following insertion into skin (2 and 10 min). Flat PS and PS-b-PAA lms were used as controls. (b) Effect of swelling time within skin on adhesion for a BCP MN adhesive with a swellable tip height fraction of 40%. (c) Representative force-displacement curve during BCP MN insertion into, and removal from pig skin. (d) Photograph of exible BCP MN adhesive (2 2 cm) prepared by using a thermoplastic elastomer as the base material (in place of PS).

(e) Adhesion on a dynamic surface. Flexible BCP MN adhesives applied to shaved skin on top of the pig wrist joint showed rm attachment during B100 cycles of bending motion. All error bars represent standard deviation.

The shape change adhesive mechanism of the BCP MN within porcine skin is shown in Fig. 3c. Following insertion into skin (Fig. 3c(i)), the tissue recoils and applies a mechanically compressive force against the BCP MN shaft. The position of the BCP MN is held xed as the inserted BCP MN immediately swells in the presence of interstitial uid and rapid volume expansion nears completion within 10 min, where the shape of the swollen tip stabilizes. The concentrated volume expansion at the tip of the BCP MN (Fig. 3c(ii)) leads to an arrow-head-like structure facilitating mechanical interlocking with the tissue. Removal of the BCP MN from the skin can be safely performed without fracture or delamination due to the deformability of the swollen tip and strong interfacial interaction between the swollen layer and base material (Fig. 3c(iii)). Signicant adhesive strength was maintained even at high levels of strain between BCP MNs and skin until BCP MNs are fully removed from the skin, indicating that the BCP MN exhibits high adhesion energy. Owing to the reversible responsiveness in water (Fig. 3c(iv)), the swollen BCP MNs quickly return to their original conical structure after removal. Following recovery of shape and stiffness (Supplementary Fig. S10), the BCP MN exhibited reversible adhesive properties during multiple swelling/deswelling cycles; therefore it is possible that the BCP MN could be reused following sterilization by ethanol or autoclave treatment.

We compared the adhesive strength of the BCP MN with currently used commercial pressure-sensitive adhesive bandages on semi-dry and wet porcine skin surfaces (Supplementary Fig. S11). While commercial bandages show higher adhesive strength on dry skin surfaces compared with the BCP MN adhesive, the adhesive strength of commercial bandages on wet skin decreased by more than 50%. However, BCP MNs showed similar adhesive strength regardless of the presence of water on the tissue surface. To demonstrate the versatility of the BCP MN adhesive, the stiff PS base material was substituted for a exible thermoplastic PS-based elastomer (Fig. 3d). The exible BCP

MN adhesive (Flex BCP MN) with 10 10 array in a 1 cm2 was

applied to a wet skin surface on top of a pig wrist joint that was cycled through an angle of B60 to stretch and compress the tissue. Flex BCP MNs maintained strong attachment without migration during 100 cycles of bending motion (Fig. 3e and Supplementary Movie 4).

MN adhesive for use in skin graft xation. With regard to potential immediate applications of BCP MN adhesives, we envision xation of skin grafts as a strong candidate. Skin grafts are often employed for closure of open wounds as a result of burns, trauma or surgical resections28,29. For successful engraftment, continuous contact between the skin graft and underlying tissue is essential to assure graft survival by directed diffusion of wound bed nutrients as well as to prevent haematoma or seroma formation30. Sutures or staples applied to the perimeter of the skin graft represent the current standard of care, and frequently this results in separation between the tissue layers, as direct xation only occurs along the periphery of the wound. Furthermore, xation by staples is not rigid enough to avoid motion and shear resulting frequently in partial or complete loss of the graft. The likelihood of failure is diminished by secondary dressings, and grafted body part immobilization.

From dye diffusion tests, we found that staples provided a surface contact area of B50% between the skin graft and underlying hydrogel, and that the staples produced signicant damage to the hydrogel during insertion and removal (Fig. 4a). In contrast, the BCP MN adhesive showed continuous contact of B100% with minimal damage to the hydrogel (Fig. 4b). As marked by an arrow in Fig. 4a, the depth of penetration (B3 mm) of staples causes signicant tissue damage and results in a higher risk of bacterial inltration. In adhesion testing, although stapled skin grafts did not fully detach from underlying tissue during pull-off, central regions of the skin grafts were separated due to

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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2715

a

b

Covered by skin graft

soaked in dye

Skin graft separation

Punctured by staples

Dye-transferred area Applied by BCP MN patch

c d

1.4

1.21.0

Force (N)

0.80.60.40.20.0

1.41.21.0

Force (N)

0.80.60.40.20.0

BCP MN patch

Continuous contact

Stapled skin graft

0 2 4 6

Distance (mm)

8 10 Seperation

12 14 16 0 2 4 6

Distance (mm)

8 10 12 14

e f

(i)

(i)

(ii)

(ii) h

Bacteria infiltrates

staple holes

Swollen microneedles

seal the holes

StaplesCyanoacrylate glue (water sealing)

Bacteria drop

g

Skin graft LB agar

Figure 4 | MN adhesive achieves effective xation of skin grafts and resists bacterial inltration. (a,b) Comparison of the contact area between a skin graft and tissue-like hydrogel (4 wt% agarose gel) after (a) applying staples and (b) a BCP MN adhesive. Staples showed less contact area between the skin graft and underlying hydrogel and on removal induced signicant damage to the underlying hydrogel (region marked by arrow in Fig. 4a), while the BCP MN adhesive showed continuous contact B100% with minimal damage to the hydrogel. (c,d) Force-displacement proles and photographs acquired during pull-off tests of skin graft on muscle tissue xed by (c) staples and (d) BCP MN adhesive. While the stapled skin graft was easily separated from the underlying muscle with a low pull-off strength, the BCP MN adhesive provided continuous contact between the skin graft and muscle tissue via mechanical interlocking with underlying muscle tissue. (eh) Comparison of the bacterial barrier property of incised skin grafts following application of (e,f) staples and (g,h) a BCP MN adhesive. (e(i)) Cartoon illustrating the primary site of bacterial inltration through the gaps between staple legs and skin grafts. (e(ii)) A photograph showing stapled skin grafts after bacteria inltration; cyanoacrylate glue was used to tightly seal the incised region (dark area outlined by dotted black line). (f) GFP-expressing E. coli colonies formed near deep staple holes (marked by red dots) where skin grafts did not appose the underlying agar layer (left, bright eld image). The inltration of E. coli through the staple holes was conrmed by green uorescence (right, uorescent image). (g(i)) Cartoon showing bacterial barrier resistance of the BCP MN adhesive resulting from tight sealing of holes by swollen MNs. (g(ii)) Photograph of the BCP MN adhesive applied on the incised skin grafts. (h) BCP adhesive prevented bacterial inltration (left, bright eld image) with minimal damage and (right, uorescent image) no green uorescence was detected on the agar plate. Scale bar, 1 mm.

low adhesion strength (Fig. 4c). Skin grafts xed using the BCP MN adhesive on muscle tissue showed signicantly higher adhesive strength (0.930.23 N cm 2) than stapled skin grafts(0.280.11 N cm 2) and non-xed skin grafts (0.220.09 N cm 2) (Table 1). The attachment was maintained following an elongation of B4 mm, which is more than ve times the height

of the BCP MN (Fig. 4d). To prevent the accumulation of uid at the interface between BCP MN adhesive and skin graft, drainage holes can easily be placed within the backing material of the adhesive array (Supplementary Fig. S12). An ideal tissue adhesive should also provide a biological barrier to reduce the risk of surgical site infection. In the case of staple xation, stress

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2715 ARTICLE

Table 1 | Tissue adhesion for skin grafts xed by BCP MN

adhesive and staples.

Fixer for skin graft Adhesion strength(N cm 2)

Work of adhesion (mJ cm 2)

0.930.23 5.231.7

Staples 0.280.11 0.60.13 Flat patch 0.220.09 0.540.26

Staples were applied at each corner of the skin graft with spacing of 1.31.5 cm, and the adhesion

strength and the work of adhesion were measured when the skin graft was separated from

the underlying muscle tissue. For the at patch control, a 12 mm diameter pin stub was afxed to

the skin graft and placed on muscle tissue without any adhesive at the interface before pull off.

Represents standard deviation from the mean (n 5).

BCP MN adhesive (10 10 MNs in

1 cm2)

concentrations localized around the staple legs can cause excessive tearing of a skin graft and produce a larger hole than a diameter of the staple (Fig. 4e). Staple holes serve as a pathway for bacterial inltration12 and as expected, Escherichia coli (E. coli) transformed to express the green uorescent protein (GFP), inltrated through the staple holes and formed colonies (Fig. 4f). For the BCP MN adhesive, the interlocked and swollen MNs tightly seal the punctured holes, thus preventing the physical passage of microorganisms (Fig. 4g). The BCP MN adhesive was removed without a signicant damage to the agar layer (left image in Fig. 4h) and GFP signal was not detected in the agar plate placed beneath the incised skin grafts covered by the BCP MN (right image in Fig. 4h). As a result of the non-permeable backing layer and tight sealing of the punctured holes by the swollen MNs, the BCP MN adhesive effectively prevents the inltration of bacteria, which is a main cause of infection.

While the micron-sized puncture marks created after removal of the MNs from the wound could serve as a source of infection, it has been reported that the skin recovers its barrier function within a few hours following MN removal31. The disappearance of puncture marks was conrmed within 1 h of removal of the swellable MN adhesive (Supplementary Fig. S13). In addition, it has been reported that microbial penetration through the holes created by MNs is minimal32. Considering its rapid and long-term adhesion, this superior bacterial barrier provided by the BCP MN adhesive could reduce the risk of infection to open wounds including burn tissue.

Firm adhesion of the MN adhesive to intestinal tissue. To evaluate the capacity for BCP MN adhesives to be used to seal intestine tissue, which may be useful to prevent leaks following gut anastomosis procedures, we examined adhesion to the outer serosal surface (Fig. 5a) that is a relatively smooth surface (roughness of several micrometres) and inner wrinkled mucosal surface (roughness on the sub-mm scale) that is covered by a sticky mucin layer (Fig. 5b). The non-swellable PS MN adhesive applied to the outer surface of the intestine tissue (Fig. 5c) showed an adhesive strength of 0.480.18 N cm 2, whereas a swellable

MN adhesive with a PS base (BCP MN) showed an adhesive strength of 1.620.17 N cm 2. Interestingly, the BCP MN adhesive exhibited an adhesive strength of 3.831.35 N cm 2 to the inner mucosal surface, possibly resulting from non-covalent interactions with mucin. Moreover, the Flex BCP MN showed further improvement in adhesive strength to the mucosal surface, up to 8 N cm 2 (mean value: 4.53 N cm 2). The enhanced adhesion of the Flex BCP MN on the mucosal surface may result from increased intimate contact during removal that may be facilitated by increased absorption of energy by the exible

backing material. The BCP MN adhesive appears to achieve adhesion to tissues that cannot be achieved effectively using conventional tape-based approaches33. As shown in Fig. 5d, inserted PS MNs into the outer surface of intestine tissue showed an average maximum torque of 0.850.28 Ncm during torsion tests, through a 60 angle of rotation. After removing PS MNs from the tissue, it was observed that B30% MNs remained broken. When the PS MNs were exposed to 100 of rotation before removal from tissue, they showed a higher maximum torque (1.220.41 Ncm) and nearly all tips or whole bodies were broken (Fig. 5e). Comparatively, less than 5% of the BCP MN and none of the Flex BCP MN were broken following 60 of rotation, and less than 5% of Flex BCP MN were broken following 100 of rotation (Fig. 5d,g). Interestingly, puncture marks resulting from the BCP MN insertion into tissue disappeared within 2 h of removal (Supplementary Fig. S14).

DiscussionThe swellable MN adhesive presented here provides a new platform for adhesion based on mechanical interlocking. It harnesses a water based swelling mechanism that responds quickly and is reversible. While we chose PS and PAA as our materials in this proof-of-concept study, the strategy described herein could be applied using many other polymers. Conventional polymer-based MN systems are composed of relatively stiff materials so that they can penetrate tissue without deformation. However, this can often lead to brittle fracture of MNs when they are subjected to dynamic tissues. As our MN system is biphasic and becomes more ductile during the swelling process, we hypothesized that our system has a distinct advantage compared with other systems in this regard. As presented here, 100% of MNs remained intact regardless of the tissue type when swollen MNs with stiff or exible cores were removed from the inserted tissues in the normal direction. In addition, the resistance of MNs to torsion-induced shear stress is compelling. Although it is difcult to quantify the exact levels of shear stress generated in this approach due to the complex interfacial geometry, at signicant levels of shear strain (induced by 60 of rotation) 100% of Flex BCP MNs remain intact, while B1/3 of PS MNs fracture.

Considering previous achievements with MN-based drug delivery, it is expected that the MN adhesive could provide a dual function enabling a rm attachment and efcient drug permeation. This strategy would be especially useful for skin graft xation, which requires intimate contact between the skin graft and the underlying wound bed over the entire surface area of the wound during movement, and a necessity to deliver medications such as anti-inammatory/anti-scarring agents, antibiotics and bioactive peptides directly to the wound bed. As drugs can easily be loaded into the swellable tips during swelling, within an agent-doped solution (followed by deswelling to return to a stiff state), BCP MN adhesive can administer multiple types of agents directly into the wound microenvironment in a minimally invasive manner. To verify the potential use of the swellable MN for delivery of drugs, we loaded BCP MN with triamcinolone acetonide (TACA), a steroid with anti-scarring and anti-inammatory properties, by allowing them to swell in a TACA solution. The BCP MN adhesive (100 MNs in 1 cm2) showed B70% release of TACA over the course of 1 day in deionized (DI) water, with the remaining TACA released over several days. Additionally, towards potential delivery of biologics, we showed that the BCP MN swellable tips could encapsulate uorescein isothiocyanate-labelled dextran (Mw: 2,000,000 Daltons) with a hydrodynamic diameter of B100 nm during the swelling process (Supplementary Fig. S15).

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a

8

c

6

* *

Adhesion strength (N cm2 )

7

6

5

4

3

2

1

0

Height (m)

4

2

0

0.0 0.2 0.4 0.6 0.8 1.0

Distance (mm)

b

500

400

Height (m)

300

200

PS MN_outside

100

PS MN_inside

BCP MN_inside

Flex BCP MN_inside

BCP MN_outside

0

0.0 0.2 0.4 0.6 0.8 1.0

Distance (mm)

d

e f g

PS MN BCP MN

Flex BCP MN

Maximum Torqueat 60 rotation (Ncm) / undamaged MNs (%)

Maximum Torqueat 100 rotation (Ncm) / undamaged MNs (%)

0.850.28 / 6615

0.480.13 / 95+

0.420.16 / None broken

1.220.41 / 167

0.790.28 / 778

0.660.32 / 95+

Figure 5 | MN adhesive rmly attaches to wet intestine tissue. (a,b) Photographs from (a) outer and (b) inner (mucosal) surfaces of pig intestine tissue and corresponding depth prole showing topographical roughness. (c) Adhesion strength for PS MN (non-swellable) and swellable BCP MN adhesives with a rigid and exible base following insertion into mucosal and serosal intestine surfaces. The asterisk indicates statistical signicance with Po0.05 (analysis of variance with post-hoc Tukeys honestly signicant difference test). Error bars represent standard deviation. (d) Following penetration into the outer surface of intestine tissue and application of 60 or 100 of rotation, signicant damage to PS MN was observed (broken MN), while BCP MN and Flex BCP MN exhibited signicantly reduced MN breakage. (eg) Photographs for tilted view of MN arrays after torsion test of 100 rotation using (e) PS MN, (f) BCP MN and (g) Flex BCP MN adhesives. The tips or entire PS MNs were broken by torsional stress (especially at the edges of the patch marked by arrow (e)), yet BCP MN with rigid and exible bases showed high resistance to torsional stress by bending in the direction of the shear force (marked by arrows in (g)).

PS and PAA were chosen as components of our swellable MN system given their established potential to exhibit only a minimal inammatory response on implantation34,35. While we envision the approach could be employed with biodegradable materials, diffusion-based drug delivery through the swollen non-degradable MN is controllable and likely useful for short-term applications.

While a change in the local acidic environment during the swelling process of PS-b-PAA could stimulate an inammatory reaction, a relatively minor change in pH (7.36.8) of PS-b-PAA is observed compared with the PAA homopolymer (7.33.4) (Supplementary Fig. S16).

As highlighted by Banga et al.36, MN-based systems for drug delivery are largely analogous to the use of hypodermic syringes, with specic advantages associated with minimal user training and lower or no risk of injury to the user and patient. While there are regulatory issues associated with medication linked to the delivery material, in our MN system the only material that remains in the tissue on removal of the MN is the loaded drug; thus reducing the potential for a chronic foreign body response.

In conclusion, we have demonstrated a new bio-inspired tissue adhesive platform governed by shape change-mediated mechanically interlocking MNs. This MN exhibits universal adhesion to soft tissues regardless of surface chemistry and it mechanically interlocks with tissues without signicant damage while providing an effective barrier to bacteria. As the BCP MN can easily

encapsulate model drugs, the MN adhesive may serve as an effective drug delivery vehicle to enhance the penetration of drugs into tissue. Owing to its capacity to conform to tissues with a broad range of surface textures, the BCP MN system has the potential for use in a wider range of medical applications than current tape-based adhesives.

Methods

Fabrication of the MN array patch. PS-b-PAA was prepared by hydrolysis of PS-b-PtBA (0.5 g, Mn: 26128 k, Polymer Source) in dry dichloromethane (20 ml) with triuoroacetic acid (1.2 molar equivalent compared with tert-butyl ester groups) as a catalyst for 48 h. Then, PS-b-PAA was precipitated into hexane, ltered, and washed several times to remove any trace of the catalyst. Hydrolysis was conrmed by NMR (Supplementary Fig. S2). The number-average-molecular weight (Mn) of

PS-b-PAA used for the swellable polymer was 26 kg mol 1 for PS and 76 kg mol 1 for PAA blocks. PS-b-PAAs with different compositions were also prepared by hydrolysis of PS-b-PtBAs (Mn: 19729 k (PDI:1.23) and 2436 k (PDI:1.09),

Polymer Source) as described above. A female PDMS mould for the MN array (10 10) of 1 cm2 was prepared using photolithography and moulding techniques

as previously reported37. Briey, to fabricate a female master mould, 1-mm-thick SU 8 lm was spin-coated onto a Si wafer and exposed to ultraviolet light (365 nm,10 W cm 2) through a chrome mask patterned with 300 mm diameter dots. The ultraviolet exposure was performed at an angle of 20 on a rotating stage at10 r.p.m. for 450 s. After developing, each conical cavity was 700 mm deep with a base diameter of 280 mm and tip radius of 10 mm. Tip-to-tip spacing between cavities was 950 mm. Female PDMS (Sylgard 184, Dow corning) moulds were obtained from male PDMS moulds cast from the female master mould. The rst layer of the MN patch was prepared by solvent casting the PS-b-PAA dissolved in DMF for 48 h followed by degassing, and the mass of material in the MN tips was controlled by altering the concentration of casting solution. To fabricate

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double-layered MN, PS pellets (Mw: 100 k, PDI: 1.06, Polysciences) were melted on solvent-casted PS-b-PAA at 180 C and then stored under vacuum for 4 h. After cooling to room temperature, double-layered MN arrays (BCP MN) were gently peeled from the mould. Single PS MN arrays were prepared by melting PS pellets in moulds at 180 C under vacuum for 4 h. To fabricate a exible MN patch (Flex BCP MN), styrene-isoprene-styrene copolymer (weight fraction of PS: 22%, Aldrich) was used in place of the PS second layer via melt casting on the underlying PS-b-PAA layer. To fabricate at PS-b-PAA substrates for adhesion testing, PS-b-PAA was solvent casted on PDMS at substrates (1 1 0.5 cm2 well) for 48 h and

then PS was lled by melt casting at 180 C for 4 h. Flat PS samples were prepared by melt casting at PDMS moulds with PS. The morphology of MN was examined with optical microscopy (E100, Nikon).

Swelling and mechanical properties of PAA homopolymer and PS-b-PAAs. PAA homopolymer (Mv: 450,000, Aldrich) and PS-b-PAAs were dissolved in DMF and cast in PDMS wells followed by drying for 7 days at room temperature in a fume hood. Casted lms with a thickness of B1 mm were cut using a 4-mm diameter biopsy punch and the punctured discs were subsequently dried at 180 C for 4 h. After cooling at room temperature, discs were immersed in phosphate-buffered saline (PBS) buffer (pH 7.4) at room temperature. The mass of each sample was measured after removal of surface water at each time point. The compressive modulus of polymer samples was characterized after they reached their maximum swollen state. The samples placed in a water bath were subjected to a compressive axial load at a strain rate of 0.5 mm min 1 using an eXpert 760 mechanical tester (ADMET) at room temperature. The load-displacement data were transformed to stress-strain plots. The slope of a linear t for 5% strain was used as a measure of the compressive modulus. The mean compressive modulus was measured from n 5 different samples.

XPS measurement to examine the surface composition of PS-b-PAA lms. To examine the surface composition of the swellable BCP MN at the PDMS (air interface following removal from the mould) and PS core interfaces, we prepared PDMS and PS substrates. For the PS-grafted surface, a hydroxyl-terminated polystyrene (PS-OH, Mn 10,000 g mol 1, PDI 1.08, Polymer Source) solution

in toluene was spin-coated onto Si wafer, cleaned by immersion for 20 min at 85 C in a piranha solution (1:4 mixture of 30% H2O2 and concentrated sulphuric acid).

The grafting of PS on the Si substrate was completed by annealing the samples at 170 C under vacuum for 1 day. Unbound PS-OHs were removed by sonication in toluene before use. The thickness of the PS brush was measured to be 78 nm by using an ellipsometer (JA Woollam company, Model WVASE32). A PS-b-PAA (26 k76 k) solution in DMF was cast on the PDMS and the PS-grafted Si wafer for 2 days and then dried at 180 C for 4 h. After cooling in air to room temperature, the prepared lms were peeled from the substrates using double-sided tape. Elemental analysis of the bottom surface of the PS-b-PAA lm that contacted the PDMS or PS brush was examined by XPS (Surface Science, model SSX-100) with a monochromatic Al Ka X-ray source.

Visualization of BCP MN shape change by swelling. To observe the swelling behaviour of the MN, one row of MNs was cut from an array attached to a metal specimen, and swelling of the MN was recorded using inverted microscope after insertion into a 1.4 wt% agarose hydrogel (Supplementary Fig. S4). Shape change of BCP MNs by swelling in animal tissue was assessed using OFDI 27, a second-generation optical coherence tomography technology. In short, light from a wavelength-swept laser source (1,2201,360 nm) was split into reference and sample arm by means of a breized interferometer. The sample light was focused into a spot with 30 mm waist radius and scanned over the tissue. The reected light was recombined with the reference signal before detection, digitized, and reconstructed to yield the depth resolved scattering prole at each scanning position. BCP MNs were inserted frontally into a cube of chicken muscle, close to the top tissue surface to enable imaging with OFDI. An area of 5 3 mm,

consisting of 1,024 512 depth proles, was scanned at a 2-min interval. The

reconstructed cross-sectional images showed signal void regions in the area of the BCP MNs, contrasted by a high scattering signal from the surrounding tissue. Minimum intensity projections along the lateral direction of MNs were taken after smoothing the tomograms to reduce speckle and provided a clear view of the shape prole of MNs.

Measurement of adhesion force with porcine skin. Full-thickness porcine cadaver skin was stored frozen and cut into B2 2 cm patches followed by

immersion in PBS for 1 h. For semi-dry adhesion tests, water was removed from the surface of porcine skin with blotting paper and the skin was dried on blotting paper for several minutes until 1020% of the absorbed water was removed. For wet adhesion testing, 200 ml of PBS buffer was spread across the surface of porcine skin before adhesion tests. Normal adhesion tests for samples with an area of 1 cm2 were performed using an eXpert 760 mechanical tester (ADMET) with custom-fabricated stainless steel tissue grips and a 50 N load cell. This methodology was selected to enable comparison with the adhesive performance of staples. A at section of skin tissue was afxed using cyanoacrylate glue to a test xture (that is, pin mount stub with diameter of 25.4 mm) and mounted within the lower grips at

the base of the mechanical tester. The MN (10 10 MNs array in 1 cm2) or at

patch were glued onto the opposing test xture and xed between the upper grips of mechanical tester. Test samples were applied to tissues with 10 N of preload at 100 mm min 1, and held in position for 2, 5, 10, 30 min or 6 h. Samples were displaced at a rate of 1 mm min 1 and the force was recorded. Dynamic adhesion testing was conducted on the shaved wrist of a pig following euthanasia. To ensure the surface was wet before testing, 1 ml of PBS buffer was spread over the skin before applying a MN patch. The degree of local skin elongation while moving the wrist from extension to maximum exion was determined. A Flex BCP MN array with 10 10 MN in 1 cm2 was adhered by applying manual compression for

10 min. The pig wrist was then subjected to dynamic testing (extension to maximum exion, back to extension) through 100 cycles to determine its xation (Supplementary Movie 4).

Measurement of contact area and adhesion force. Unmeshed porcine skin grafts with a thickness of 200 mm were obtained from the back of postmortem pigs using a dermatome immediately after euthanasia, in accordance with animal health and welfare policies. They were kept in Dulbeccos modied Eagle medium (DMEM) at 4C and used within 2 weeks of harvest. To examine the contact area between the adhesive patch and underlying tissue, dye diffusion tests were performed. After application of the MN patch or staples, the skin graft (1.5 1.5 cm)

was presoaked in 0.1 wt% solution of Rhodamine dye for 10 min and transferred onto a 4 wt% agarose hydrogel after removal of the excess dye solution. Staples or a BCP MN adhesive were applied to x the skin graft onto the hydrogel for 2 min. The contact area was determined by the dye-transferred region via image analysis software. For adhesion tests, skin grafts were cut into 1.5 1.5 cm patches and

placed on a at section of muscle tissue (2.5 2.5 cm) that had been afxed using

cyanoacrylate glue to the bottom test xture. The MN patches (10 10 MNs array

in 1 cm2) were glued to the top xture of the mechanical tester and applied to the underlying tissues with 20 N of preload at 150 mm min 1 and held in position for 10 min. Samples were then displaced at a rate of 1 mm min 1 and the force was recorded. To compare adhesion force between the stapled skin graft and underlying muscle tissues, staples were applied to the perimeter of the skin graft with spacing of 1.31.5 cm usinga skin stapler (Reex One, CONMED). For experiments with the MN patch, the same preload (20 N) was used and a pin mount stub with a diameter of 12 mm was applied to the stapled skin graft. For the duration of all experiments, the tissue was kept moist with PBS.

In vitro assessment of bacterial inltration. BL21(DE3) competent E. coli (New England Biolabs) was transformed with pFluoroGreen (EDVO-Kit 223, EDVOTEK) and cultured on ampicillin and isopropyl-b-D-thiogalactoside (IPTG)

supplemented LB agar plates. A GFP-positive colony was picked to inoculate a 10 ml standard LB broth supplemented with ampicillin and IPTG. The GFP-expressing E. coli was cultured overnight at 37 C and was diluted by 103-fold into PBS buffer. This dilution yielded an inoculum of B2 104 c.f.u. ml 1. To prepare

LB agar plates, LB agar medium powder (MP Biomedicals, LLC) was added to DI water and autoclaved at 121 C for 15 min. After cooling to 50 C, 15 ml of molten agar was supplemented with ampicillin and IPTG, poured into sterile Petri dishes (100 15 mm) and allowed to solidify. A 50 ml solution of bacteria (B1,000

cells) was pipetted onto the centre of the incised skin grafts (250 mm thick) placed on LB agar plate after applying staples or the BCP MN adhesive. To investigate the effect of staple holes on bacteria inltration, the interface of two incised skin grafts was tightly sealed using cyanoacrylate glue before stapling. Testing plates were incubated at 37 C for 24 h and were observed for the growth of GFP-expressingE. coli to examine the bacterial barrier resistance of the incised skin grafts closed by a BCP MN adhesive or staples (n 3).

Measurement of adhesion and torsional force with porcine small intestine. Fresh intestine tissue was rinsed with PBS buffer several times and then cut intoB2 2 cm patches. Surface water was removed with blotting paper while

mucin remained on the inner surface of the intestine. Topography for the inner and outer surface of intestine tissue was measured by using a depth proler (Sloan Dektak II) after freezing the tissue at 20 C. Normal adhesion tests were con

ducted using the same experimental setup for porcine skin. For the duration of all experiments, the tissue was kept moist with PBS. The mean adhesion force was measured from n 5 different samples. Torsion tests were conducted using a

biaxial transducer followed by the same experimental procedures used for the normal adhesion test. Samples inserted into tissue were rotated at a rate of 0.5 degree s 1 by 60 or 100 degree, and the force was recorded. Following the torsion test, breakage of MN was examined via macroscopic images.

Controlled release of drugs from swellable tips of BCP MN adhesive. Triamcinolone acetonide (TACA, Fluka) was loaded in the tips of BCP MN patch (100 MNs in 1 cm2) via swelling in a TACA solution (4 ml) of 1 mg ml 1 dissolved in methanol. Following 30 min incubation, the BCP MN patch was washed by dipping in DI water, excess water on the surface of the needles was removed, and then the patches were dried at room temperature for 1 h. Drug-loaded BCP MN patches were placed into 6 ml of fresh DI water to examine the release of

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encapsulated TACA. A quantity of 0.6 ml of the solution was sampled at each time point, and 0.6 ml of the fresh DI water was replaced. The amount of released TACA was determined by HPLC system (Agilent 1100 series) with C18 column (5 mm)

(250 4.6 mm ID, Agilent Eclipse XDB-C18) at 240 nm with an Agilent G1314A

detector. Peak identication was achieved based on the comparison of retention times of compounds within standard solutions using ChemStation software (Agilent Technologies).

Statistics. Unless otherwise stated, all experiments were performed using at least ve samples per group and the data presented are representative of ve independent experiments. For multiple comparisons, analysis of variance was performed with the Tukeys honestly signicant difference test at signicance levels of 95%. Error bars in bar graphs represent the standard deviation.

References

1. Majumder, A., Ghatak, A. & Sharma, A. Microuidic adhesion induced by subsurface microstructures. Science 318, 258261 (2007).

2. Karp, J. M. & Langer, R. Dry solution to a sticky problem. Nature 477, 4243 (2011).

3. Vote, B. J. & Elder, M. J. Cyanoacrylate glue for corneal perforations: a description of a surgical technique and a review of the literature. Clin. Experiment. Ophthalmol. 28, 437442 (2000).

4. Lauto, A., Mawad, D. & Foster, L. J. R. Adhesive biomaterials for tissue reconstruction. J. Chem. Technol. Biotechnol. 83, 464472 (2008).

5. Artzi, N., Shazly, T., Baker, A. B., Bon, A. & Edelman, E. R. Aldehyde-amine chemistry enables modulated biosealants with tissue-specic adhesion. Adv. Mater. 21, 33993403 (2009).

6. Strehin, I., Nahas, Z., Arora, K., Nguyen, T. & Elisseeff, J. A versatile pH sensitive chondroitin sulfatePEG tissue adhesive and hydrogel. Biomaterials 31, 27882797 (2010).

7. Hida, K. et al. Nonsuture dural repair using polyglycolic acid mesh and brin glue: clinical application to spinal surgery. Surg. Neurol 65, 136142 (2006).

8. Cimador, M., Castagnetti, M., Milazzo, M., Sergio, M. & Grazia, E. D. Suture materials: do they affect stula and stricture rates in ap urethroplasties? Urol. Int. 73, 320324 (2004).

9. Murray, K. D., Ho, C.-H., Hsia, J.-Y. J. & Little, A. G. The inuence of pulmonary staple line reinforcement on air leaks. Chest 122, 21462149 (2002).

10. Rosser, J. C., Rosser, L. E. & Savalgi, R. S. Skill acquisition and assessment for laparoscopic surgery. Arch. Surg. 132, 200204 (1997).

11. Ferzli, G. S., Frezza, E. E., Pecoraro, A. M. & Ahern, K. D. Prospective randomized study of stapled versus unstapled mesh in a laparoscopic preperitoneal inguinal hernia repair. J. Am. Coll. Surg. 188, 461465 (1999).

12. Smith, T. O., Sexton, D., Mann, C. & Donell, S. Sutures versus staples for skin closure in orthopaedic surgery: meta-analysis. Br. Med. J. 340, 18 (2010).

13. Kim, J. & Stevenson, T. R. Abdominoplasty, liposuction of the anks, and obesity: analyzing risk factors for seroma formation. Plast. Reconstr. Surg. 117, 773779 (2006).

14. Pepicello, J. & Yavorek, H. Five year experience with tape closure of abdominal wounds. Surg. Gynecol. Obstet. 169, 310314 (1989).

15. Harold, K. L. et al. Optimal closure method of ve-millimeter trocar sites. Am.J. Surg. 187, 2427 (2004).16. Coulthard, P. et al. Tissue adhesives for closure of surgical incisions. Cochrane Database Syst. Rev. 5, CD004287 (2010).

17. Hollinsky, C. et al. Comparison of a new self-gripping mesh with other xation methods for laparoscopic hernia repair in a rat model. J. Am. Coll. Surg 208, 11071114 (2009).

18. Hammond, R. A. The proboscis mechanism of Acanthocephalus Ranae. J. Exp. Biol. 45, 203213 (1966).

19. Reed, M. L., Han, H. & Weiss, L. E. Silicon micro-velcro. Adv. Mater. 4, 4851 (1992).

20. Mori, H. & Muller, A. H. E. New polymeric architectures with (meth)acrylic acid segments. Prog. Polym. Sci. 28, 14031439 (2003).

21. Xu, C. et al. Reversible stimuli-responsive nanostructures assembled from amphiphilic block copolymers. Nano Lett. 6, 282287 (2006).

22. Frster, S. & Antonietti, M. Amphiphilic block copolymers in structure-controlled nanomaterial hybrids. Adv. Mater. 10, 195217 (1998).

23. Weidisch, R. et al. A novel scheme for prediction of deformation mechanisms of block copolymers based on phase behavior. Macromolecules 34, 25282535 (2001).

24. Arndt, K. F. et al. Poly(vinyl alcohol)/poly(acrylic acid) hydrogels: FT-IR spectroscopic characterization of crosslinking reaction and work at transition point. Acta Polym. 50, 383390 (1999).

25. Nagarajan, S., Russell, T. P. & Watkins, J. J. Dual-tone patterned mesoporous silicate lms templated from chemically amplied block copolymers. Adv. Funct. Mater. 19, 27282734 (2009).

26. Sandby-Moller, J., Poulsen, T. & Wulf, H. C. Epidermal thickness at different body sites: relationship to age, gender, pigmentation, blood content, skin type and smoking habits. Acta Derm. Venereol. 83, 410413 (2003).

27. Yun, S., Tearney, G., de Boer, J., Iftimia, N. & Bouma, B. High-speed optical frequency-domain imaging. Opt. Express 11, 29532963 (2003).

28. Currie, L. J., Sharpe, J. R. & Martin, R. The use of brin glue in skin grafts and tissue-engineered skin replacements: a review. Plast. Reconstr. Surg. 108, 17131726 (2001).

29. Iwuagwu, F. C., Wilson, D. & Bailie, F. The use of skin grafts in postburn contracture release: a 10-year review. Plast. Reconstr. Surg. 103, 11981204 (1999).

30. Gibran, N. et al. Comparison of brin sealant and staples for attaching split-thickness autologous sheet grafts in patients with deep partial-or full-thickness burn wounds: a phase 1/2 clinical study. J. Burn Care Res. 28, 401408 (2007).

31. Gupta, J., Gill, H. S., Andrews, S. N. & Prausnitz, M. R. Kinetics of skin resealing after insertion of microneedles in human subjects. J. Control. Release 154, 148155 (2011).

32. Donnelly, R. et al. Microneedle arrays allow lower microbial penetration than hypodermic needles in vitro. Pharm. Res. 26, 25132522 (2009).

33. Kwak, M. K., Jeong, H.-E. & Suh, K. Y. Rational design and enhanced biocompatibility of a dry adhesive medical skin patch. Adv. Mater. 23, 39493953 (2011).

34. Chauvel-Lebret, D. J., Pellen-Mussi, P., Auroy, P. & Bonnaure-Mallet, M. Evaluation of the in vitro biocompatibility of various elastomers. Biomaterials 20, 291299 (1999).

35. De Giglio, E. et al. Biocompatibility of poly(acrylic acid) thin coatings electro-synthesized onto TiAlV-based implants. J. Bioact. Compat. Polym. 25, 374391 (2010).

36. Banga, A. K. Microporation applications for enhancing drug delivery. Expert Opin. Drug Deliv. 6, 343354 (2009).

37. Park, J.-H., Choi, S.-O., Seo, S., Choy, Y. B. & Prausnitz, M. R. A microneedle roller for transdermal drug delivery. Eur. J. Pharm. Biopharm. 76, 282289 (2010).

Acknowledgements

We would like to thank ADMET for providing an eXpert 760 mechanical tester that was

instrumental for this work. Funding for this research was provided by the National

Institutes of Health (NIH) grant GM086433 to J.M.K., NIH grant P41EB015903 to B.E.B.

and the Basic Science Research Program through the National Research Foundation

of Korea (NRF) funded by the Ministry of Education, Science and Technology

(2012R1A6A3A03039055). This work was performed in part through the use of MITs

Microsystems Technology Laboratories and Harvards Center for Nanoscale Systems. We

would like to thank Albert Kwon for providing E. coli and Dr. Bryan Laulicht for helpful

discussions.

Author contributions

S.Y.Y. and J.M.K. conceptualized and directed the research project. S.Y.Y., J.M.K. and

E.D.O. prepared the manuscript. S.Y.Y., E.D.O., G.C.S., K.M.P., W.K.C., M.V., B.E.B. and

B.P. performed the experiments and data analysis with J.M.K. All authors discussed the

results and commented on the manuscript.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Web End =naturecommunications Competing nancial interests: The authors declare no competing nancial interests. Reprints and permission information is available online at http://npg.nature.com/reprintsandpermissions/

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How to cite this article: Yang, S.Y. et al. A bio-inspired swellable microneedle adhesive

for mechanical interlocking with tissue. Nat. Commun. 4:1702 doi: 10.1038/ncomms2715

(2013).

10 NATURE COMMUNICATIONS | 4:1702 | DOI: 10.1038/ncomms2715 | http://www.nature.com/naturecommunications

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