Abstract

   Tissue regeneration is regulated by morphological clues of implants in
   bone defect repair. Engineered morphology can boost regenerative
   biocascades that conquer challenges such as material bioinertness and
   pathological microenvironments. Herein, a correlation between the liver
   extracellular skeleton morphology and the regenerative signaling,
   namely hepatocyte growth factor receptor (MET), is found to explain the
   mystery of rapid liver regeneration. Inspired by this unique structure,
   a biomimetic morphology is prepared on polyetherketoneketone (PEKK) via
   femtosecond laser etching and sulfonation. The morphology reproduces
   MET signaling in macrophages, causing positive immunoregulation and
   optimized osteogenesis. Moreover, the morphological clue activates an
   anti‐inflammatory reserve (arginase‐2) to translocate retrogradely from
   mitochondria to the cytoplasm due to the difference in spatial binding
   of heat shock protein 70. This translocation enhances oxidative
   respiration and complex II activity, reprogramming the metabolism of
   energy and arginine. The importance of MET signaling and arginase‐2 in
   the anti‐inflammatory repair of biomimetic scaffolds is also verified
   via chemical inhibition and gene knockout. Altogether, this study not
   only provides a novel biomimetic scaffold for osteoporotic bone defect
   repair that can simulate regenerative signals, but also reveals the
   significance and feasibility of strategies to mobilize
   anti‐inflammatory reserves in bone regeneration.

   Keywords: arginase‐2, biomimetic surface modification, macrophage
   metabolic reprogramming, osseointegration, polyetherketoneketone
     __________________________________________________________________

   Bioinspired by the liver extracellular skeleton morphology, a
   functional polyetherketoneketone scaffold is prepared that reproduces
   similar regenerative signaling, causing positive immunoregulation and
   osteogenesis. The morphology activates an anti‐inflammatory reserve
   (arginase‐2) to translocate retrogradely from mitochondria to the
   cytoplasm, reprogramming metabolism of energy and arginine. This study
   provides a novel biomimetic scaffold and an effective strategy for
   osteoporotic bone defect repair.

   graphic file with name ADVS-10-2302136-g005.jpg

1. Introduction

   Currently, designing efficient strategies to repair bone defects caused
   by trauma and diseases still faces enormous challenges.^[ [46]^1 ^] In
   the field of tissue engineering, the application of bone substitute
   materials, such as biological scaffolds, represents a promising
   approach to repairing defects.^[ [47]^2 ^] Compared with metal or
   ceramic materials, polyetherketoneketone (PEKK) is considered a
   desirable candidate for bone tissue engineering due to its excellent
   mechanical properties, which are more consistent with the prerequisites
   for biological scaffolds.^[ [48]^3 ^] However, its inherent
   bioinertness impairs physiological regenerative biocascades, leading to
   intense inflammatory responses and osteoblast dysfunction.^[ [49]^4 ^]
   Therefore, a deliberate strategy needs to be designed to overcome the
   limitations.

   To date, strategies to modify PEKK, a member of polyaryletherketones
   (PAEKs), have mainly focused on adding bioactive particles to a
   prepared porous surface to steer cell fates.^[ [50]^5 ^] Nevertheless,
   concerns, including burst release and effective dose deviation, require
   attention.^[ [51]^6 ^] Thus, the concept of tissue‐inducing
   biomaterials has been introduced, defined as a biomaterial designed to
   induce the regeneration of damaged tissue without adding exogenous
   cells or bioactive factors.^[ [52]^7 ^] However, conventional
   modification designs on PEKK and other similar PAEKs by constructing
   porous structures using sulfonation have not been successful to
   orchestrate regenerative biocascades.^[ [53]^3 , [54]^8 ^] The
   morphological clues regulating tissue regeneration need to be further
   explored.

   Incorporating the wisdom of the natural morphological clues of
   organisms, biomimetic strategies have shown unique superiority in the
   design of scaffold materials for tissue repair.^[ [55]^5 , [56]^9 ^]
   However, solely imitating the physical morphology and disregarding the
   transformation of intrinsic biological signals is insufficient. It is
   of greater significance to reproduce the corresponding biological
   functions to achieve functional bionics via the recurrence of
   morphological clues. Among natural tissues, liver tissue exhibits
   outstanding regenerative ability. Many growth factors, most notably
   hepatocyte growth factor (HGF), play important roles in tissue
   regeneration.^[ [57]^10 ^] MET, known as the HGF receptor, is required
   for various morphogenetic bioevents and promotes tissue remodeling,
   such as bone regeneration, which underlies wound repair and organ
   homeostasis.^[ [58]^11 ^] MET signaling also plays a crucial role in
   the organization and transformation of external signals into
   intracellular biochemical information.^[ [59]^11 ^] In addition to its
   multistage blood supply, the extracellular skeleton structure of the
   liver may explain the rapid immune response and tissue repair.^[
   [60]^12 ^] These findings suggest a potential synergistic relationship
   between the liver skeleton structure and MET signaling, which is worth
   studying and applying to improve bone tissue repair.

   Macrophages, as pioneers of the immune response and regeneration, can
   modulate the microenvironment in response to morphological clues on the
   implanted material surface to pave the way for subsequent tissue
   repair.^[ [61]^13 ^] Material bioinertness and improper morphological
   clues will disrupt regenerative immune orchestration, leading to
   delayed bone healing.^[ [62]^14 ^] The negative effect is particularly
   evident in complicated pathological microenvironments, such as
   osteoporosis.^[ [63]^15 ^] Yet osteoporosis poses higher requirements
   for optimal mechanical properties, deepening the contradiction between
   the need for and concerns about PEKK application.^[ [64]^16 ^] In
   addition, a prolonged inflammatory and pathological milieu can weaken
   the original anti‐inflammatory systems, such as arginase‐1 (Arg1),
   attenuating the effect of exogenous immunoregulation.^[ [65]^17 ^]
   Arginase‐2 (Arg2), different from Arg1, can serve as a reserve and
   reprogram arginine metabolism in an inflammatory microenvironment to
   resurge macrophage state.^[ [66]^18 ^] Moreover, macrophages are
   modulated by MET signaling, which is influenced by designated
   morphological clues and can be pertinently reprogrammed to orchestrate
   immunoregulation and tissue regeneration.^[ [67]^11 ^] Given this, an
   engineered design derived from liver morphological clues and a rational
   utilization of Arg2 can endow PEKK with biological regulation ability
   and revitalize its clinical application.

   Herein, we found that the liver extracellular skeleton structure could
   effectively activate MET signaling to regulate macrophage polarization.
   Inspired and guided by this unique morphological bio‐template, a
   biomimetic surface structure was designed and prepared on PEKK
   scaffolds via femtosecond laser etching and sulfonation. The biomimetic
   design reproduced MET signaling in macrophages and conferred bioinert
   PEKK with biological functionalization, orchestrating regenerative
   immunoregulation and optimizing osteogenesis. MET signaling of
   biomimetic morphology mediates retrograde translocation of Arg2 from
   mitochondria into the cytoplasm, reprogramming energy metabolism and
   arginine metabolism (Scheme [68]1 ). This deliberate strategy
   incorporates morphological signaling transduction and mobilization of
   the host's anti‐inflammatory reserves, showing great promise in
   overcoming the barriers of material bioinertness and scaffold
   application in an inflammatory microenvironment.

Scheme 1.

   Scheme 1
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   Schematic diagram showing the biomimetic functionalization design for
   liver‐inspired PEKK scaffolds and the mechanism of macrophage metabolic
   reprogramming in osteoporotic bone regeneration via mobilization of
   anti‐inflammatory reserves.

2. Results

2.1. MET Signaling Induced by the Liver Extracellular Skeleton Structure

   MET signaling is involved in functional cooperation between different
   signal axes and pathways related to macrophage regenerative
   immunoregulation.^[ [70]^11 ^] When HGF activated MET signaling,
   RAW264.7 cells exhibited M2 polarization (Figure [71]1A). However, this
   change was eliminated by the MET inhibitor (SGX‐523), indicating a
   strong correlation between MET signaling and M2 polarization. To
   explore the relationship between morphological clues and biological
   information in tissue regeneration, the liver extracellular skeleton
   structures were prepared. After decellularization (Figure [72]S1A–C,
   Supporting Information) and lyophilization, the surface of the liver
   tissue blocks presented a 3D petal‐like unit morphology with an
   interval of ≈6 µm (denoted as “Skeleton”) (Figure [73]1B). For
   Skeleton‐Flat, the acellular tissue blocks were physically extruded
   prior to lyophilization to remove surface characteristic structures
   without affecting their composition (Figure [74]S1D,E, Supporting
   Information). Interestingly, the characteristic structure increased
   p‐MET expression (Figure [75]1C) and promoted early immune repair
   transformation, showing higher CD206 (M2) and lower inducible nitric
   oxide synthase (iNOS, M1) expression (Figure [76]1D). Blockage of MET
   (using a MET antibody) eliminated the polarization difference between
   macrophages on the two skeleton samples, indicating that the unique
   structure mediated M2 polarization via MET signaling. It is noteworthy
   that macrophages on the surface of the liver skeleton still showed a
   preference for M2 polarization when HGF was neutralized (using HGF
   antibody), compared with those on the surface without the
   characteristic structure (Figure [77]1D). These findings suggested that
   the characteristic structure could simulate MET signaling and realize
   the transformation of morphological clues into biological information.
   Furthermore, compared to titanium, PEKK scaffolds were unable to
   increase the levels of MET signaling and M2 polarization of macrophages
   (Figure [78]1E,F), which might explain the intrinsic cause of PEKK
   bioinertness. The release of osteogenic factors by macrophages was also
   inhibited (Figure [79]1G,H). From this, we propose that one peculiar
   avenue to endow PEKK with biological regulation ability is to harness
   the morphological resources of the liver.

Figure 1.

   Figure 1
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   MET signaling triggered by the liver extracellular skeleton. A) Heat
   map depicting the expression of genes related to polarization of
   RAW264.7 cells stimulated by hepatocyte growth factor (HGF) and the MET
   inhibitor (SGX‐523) (n = 3; *, #, and $ represent HGF vs control, HGF
   vs HGF+SGX‐523, and HGF+SGX‐523 vs control). B) Scanning electron
   microscopy (SEM) images capturing surface of the Skeleton sample and
   the Skeleton‐Flat sample. C) Expression of MET and p‐MET of RAW264.7
   cells cultured on the Skeleton sample and the Skeleton‐Flat sample. D)
   Immunofluorescent staining images of polarization markers of RAW264.7
   cells cultured on two liver extracellular skeleton samples treated with
   HGF antibody or MET antibody for 3 days (iNOS, green; CD206, red; DAPI,
   blue). E) Expression of MET and p‐MET of RAW264.7 cells cultured on the
   PEKK scaffold and the titanium scaffold for 3 days. F) Heat map
   depicting the expression of genes related to polarization of RAW264.7
   cells cultured on the PEKK scaffold and the titanium scaffold relative
   to that of the control group treated with or without SGX‐523 (n = 3; *,
   ^, #, and $ represent PEKK vs Ti, PEKK+SGX‐523 vs Ti+SGX‐523, PEKK vs
   PEKK+SGX‐523, and Ti vs Ti+SGX‐523, respectively). The concentrations
   of G) BMP2 and H) TGF‐β of the microenvironment regulated by RAW264.7
   cells cultured on the PEKK scaffold and the titanium scaffold for 4
   days (error bars, means ± SD; n = 5). Data were analyzed by ordinary
   one‐way ANOVA with Tukey's post‐hoc test and respective P values are
   provided.

2.2. Preparation and Characterization of Biomimetic PEKK Scaffolds

   Bioinspired by the liver extracellular skeleton structure and guided by
   this bio‐template, we constructed the characteristic structure by
   physical and chemical means. As shown in Figure [81]2A, the palisade
   structures (8 µm apart) were prepared by femtosecond laser (Figure
   [82]S2A, Supporting Information), which were further processed with 80%
   H[2]SO[4] to create an independent petal‐like unit morphology on the
   PEKK surface (denoted as “PEKK‐L”), with hydrothermal treatment
   subsequently to remove residual acid. As a mainstream means of
   modification, sulfonating PEKK with 98% H[2]SO[4] was included as a
   comparison (PEKK‐SW) in this study.^[ [83]^19 ^] The 3D petal‐shaped
   unit morphology of the PEKK‐L surface resembled the natural
   extracellular skeleton of the liver, which was different from the
   smooth surface and the microporous network of the PEKK and PEKK‐SW
   surfaces, respectively (Figure [84]2B). The lamellar prominences of the
   PEKK‐L surface were closely spaced ≈5 µm apart (Figure [85]2B), and the
   biomimetic modified layer was ≈30 µm thick (Figure [86]S2B, Supporting
   Information). The unique morphology with microgrooves, islands, and
   microholes on the PEKK‐L surface was similar to that of the liver
   skeleton rather than the flat or single porous structure on the PEKK
   and PEKK‐SW surfaces (Figure [87]2C). PEKK‐L shared a semblable texture
   and undulating shape with the liver skeleton (Figure [88]2D). In
   addition, the surface roughness of the PEKK‐L (≈0.884 µm) was close to
   that of the liver skeleton (≈0.844 µm) (Figure [89]2E). The composition
   of PEKK was not significantly disturbed by the addition of a small
   amount of exogenous sulfur (only 1.08%) (Figure [90]S2C, Supporting
   Information). The asymmetric tensile vibration peak of O=S=O was at
   1255 cm^−1 in the FTIR spectra (Figure [91]2F), indicating the
   formation of —SO[3]H groups.^[ [92]^20 ^] Moreover, the characteristic
   peak at 1100 cm^−1 was attributed to C—OH groups on the PEKK‐L surface,
   resulting from the C—O—C bond breaking and reacting with water
   molecules to form C—OH groups during the laser etching process.^[
   [93]^21 ^] The introduction of hydrophilic components (—SO[3]H groups)
   and active sites (—OH groups) is expected to improve the hydrophilicity
   of PEKK, considering the combined effects of these functional groups,
   as well as surface roughness and morphological clues, which can
   significantly influence the contact angle (Figure [94]S2D, Supporting
   Information).^[ [95]^5 , [96]^21 , [97]^22 ^] The pH value remained
   largely unaffected by the introduction of the functional groups (Figure
   [98]S2E, Supporting Information). Fortunately, PEKK‐L maintained the
   prominent mechanical properties of polymer materials, which possessed
   an appropriate elastic modulus (≈10.469 GPa) matching that of natural
   bone tissue (Figure [99]2G).^[ [100]^23 ^] The unique morphology and
   outstanding mechanical properties of PEKK‐L make it a promising
   candidate for use in biomedical engineering.

Figure 2.

   Figure 2
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   Preparation and characterization of biomimetic PEKK scaffolds. A)
   Schematic illustration of the preparation of PEKK‐L scaffolds. B)
   Scanning electron microscopy (SEM) images, C) 3D surface optical
   profiles and D) atomic force microscopy (AFM) images of various PEKK
   scaffolds and the liver extracellular skeleton. E) Surface roughness of
   the PEKK scaffolds and the liver extracellular skeleton detected by 3D
   optical profiles (error bars, means ± SD; n = 4). F) Fourier transform
   infrared (FT‐IR) spectra of various PEKK scaffolds. G) Elastic modulus
   of various PEKK scaffolds and the liver extracellular skeleton detected
   by AFM (error bars, means ± SD; n = 3) with elastic modulus of
   representative natural bone tissue according to references. Statistical