Multiscale osteointegration as a new paradigm for the design of calcium phosphate scaffolds for bone regeneration

Biomaterials 31 (2010) 3552–3563

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Multiscale osteointegration as a new paradigm for the design of calcium phosphate scaffolds for bone regeneration Sheeny K. Lan Levengood a,1, Samantha J. Polak b, Matthew B. Wheeler c, Aaron J. Maki b, Sherrie G. Clark d, Russell D. Jamison e, Amy J. Wagoner Johnson f, * a

Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, 1304 West Green Street, Urbana, IL 61801, USA Department of Bioengineering, University of Illinois at Urbana-Champaign, 1304 West Springfield Avenue, Urbana, IL 61801, USA Department of Animal Sciences, University of Illinois at Urbana-Champaign, 1207 West Gregory Drive, Urbana, IL 61801, USA d Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, 1008 West Hazelwood Drive, Urbana, IL 61802, USA e School of Engineering, Virginia Commonwealth University, 601 West Main Street, Suite 331, Richmond, VA 23284-3068, USA f Department of Mechanical Science and Engineering, University of Illinois at Urbana-Champaign, 1206 West Green Street, Urbana, IL 61801, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 October 2009 Accepted 12 January 2010 Available online 11 February 2010

The role of macropore size (>100 mm) and geometry in synthetic scaffolds for bone regeneration has been studied extensively, but successful translation to the clinic has been slow. Significantly less attention has been given to porosity at the microscale (0.5–10 mm). While some have shown that microporosity in calcium phosphate (CaP)-based scaffolds can improve rate and extent of bone formation in macropores, none has explored microporosity as an additional and important space for bone ingrowth. Here we show osteointegration of biphasic calcium phosphate (BCP) scaffolds at both the macro and micro length scales. Bone, osteoid, and osteogenic cells fill micropores in scaffold rods and osteocytes are embedded in mineralized matrix in micropores, without the addition of growth factors. This work further highlights the importance of considering design parameters at the microscale and demonstrates the possibility for a bone–scaffold composite with no ‘‘dead space.’’ Embedded osteocytes distributed throughout microporous rods may form a mechanosensory network, which would not be possible in scaffolds without microporosity. Multiscale osteointegration has the potential to greatly improve overall performance of these scaffolds through an improvement of mechanical properties, load transfer, and stability in the long and short term, and represents a new paradigm for scaffold design. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Bone ingrowth Calcium phosphate Porosity Microstructure Osteointegration

1. Introduction The prevailing paradigm in the engineering of porous materials, or scaffolds, for bone defect repair has been to focus primarily on optimizing the scaffold architecture. The elusive goal is for architecture, and by extension the scaffold porosity, to meet mechanical and transport needs, while also maximizing rate and extent of bone ingrowth for a wide range of defect shapes and sizes [1–5]. Some attention has been given to scaffold design at the centimeter scale to address anatomically complex defects [6–8], but significantly more attention has been paid to the scale of scaffold macroporosity (i.e. >100 mm) [1,3–5]. Pore sizes reported to support osteointegration range from 200 to1600 mm in a variety of materials and pore * Corresponding author. Tel.: þ1 217 265 5581; fax: þ1 217 244 6534. E-mail address: [email protected] (A.J. Wagoner Johnson). 1 Present address: 3153 Engineering Centers Building, 1550 Engineering Drive, Madison, WI 53706, USA. 0142-9612/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2010.01.052

configurations [2]. In contrast to macroporosity, less attention has been given to design at the level of the scaffold material microporosity, which is typically defined as 0.5–10 mm. Given the hierarchical structure of bone [9,10], engineering scaffolds to promote and sustain bone ingrowth over all of these length scales (cm to <10 mm) merits attention. Microporosity in the struts or walls of macroporous calcium phosphate (CaP) scaffolds has been identified as an important scaffold component but, to date, the focus has been on the presence of microporosity enhancing bone formation in macropores. This work exploits both macroporosity and microporosity in CaP bone tissue engineering scaffolds for the specific purpose of achieving multiscale osteointegration. CaPs have been used in a range of orthopedic and dental applications [11–14] and have been investigated for use as bone tissue engineering scaffolds [1,15–17] because of their inherent bioactivity (forms chemical bond with bone) [18] and osteoconductivity (supports bone growth) [19]. Of the CaPs, a mixture of hydroxyapatite (HA) and b-tricalcium phosphate (b-TCP), or

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biphasic calcium phosphate (BCP), may be the best candidate material for use as bone grafts. Both phases are important for the success of a BCP scaffold in promoting bone regeneration. HA is more stable, as the dissolution rate is much lower, resulting in strength retention [19–21]. b-TCP, on the other hand, is more soluble and its degradation products, Ca2þ and PO3 4 , are important for promotion of bioactivity [18,22]. The porous CaP scaffolds of interest here are fabricated via a solid freeform fabrication (SFF) method, micro-robotic deposition (mRD) [23]. Hollister asserts that scaffolds with ‘‘designed architectures,’’ or scaffolds made using SFF methods that allow for strict architectural control, can better meet the mechanical requirements for hard tissues as compared to scaffolds fabricated by more traditional methods [2]. mRD allows for control over both macro- and microporosity and there is evidence that these CaP-based bone scaffolds with multiscale porosity may have the appropriate strength and stiffness for use in load-bearing applications [15,24–27]. We and others have shown that microporosity in CaP scaffolds can enhance the rate and extent of bone regeneration within macropores [27–30] and can also promote bone formation in ectopic sites [27,31]. However, in these studies, microporosity was used as a mechanism to enhance scaffold osteoconductivity or elicit scaffold osteoinductivity [29,32,33]. While some suggest that bone can grow into micropores immediately adjacent to scaffold macropores [30,34,35], we focus on microporosity as important space for bone growth and show growth throughout scaffold struts or rods. To our knowledge, this paper is the first to show multiscale osteointegration for any bone tissue engineering scaffold and this paper focuses specifically on characterization of bone within micropores of biphasic calcium phosphate (BCP) scaffolds that also contain macroporosity. 2. Materials and methods 2.1. Scaffold fabrication and characterization Commercially available hydroxyapatite (HA) powder (Riedel-de Haen, Seelze, Germany) was previously characterized by Hoelzle and Cordell [23,36] and slurries were prepared according to the methods of Dellinger et al. and Michna et al. [37,38]. Briefly, as-received HA powder was calcined at 1100  C to reduce its surface area and enhance its processability. During calcination, the powder was heated at 1000  C/h to 1100  C, held for 10 h, and subsequently cooled to ambient temperature. Calcined powder was ball-milled with alumina media to reduce the average particle size and break up agglomerates. The HA ink suspensions were prepared by first mixing appropriate amounts of deionized water and Darvan 821A (R.T. Vanderbilt, Norwalk, CT). The solution pH was adjusted to 10 by the addition of 5 M NH4OH after which the HA powder was added to the solution. The suspension was mixed on a paint shaker and sonicated, then centrifuged for 60 min to concentrate it. After decanting the supernatant, the concentrated HA was homogenized by mixing again using the paint shaker. Appropriate amounts of polymethylmethacrylate (PMMA) microspheres (Matsumoto Microsphere M-100, Tomen American, New York, NY) that served as fugitive porogens, water, Methocel and 1-octanol were added to reach a solids loading of 55% and the suspension mixed again. The pH of the suspension was adjusted by the incremental addition of 1 M HNO3 as needed, which modified the viscocity. A poly(ethyleneimine) (PEI) gelling agent was added once the appropriate viscocity had been reached. Rectangular lattices with alternating layers of orthogonal rods were fabricated using micro-robotic deposition [23]. Lattices were next heated incrementally to 900  C to burn out organic binders and PMMA, sintered at 1300  C for 2 h, then furnace-cooled to ambient temperature. While the as-received powder was 100% HA, the material underwent a phase transformation during sintering leading to a final composition of 87% HA and 13% b-TCP, verified using X-ray diffraction (XRD). This combination of phases is referred to as biphasic calcium phosphate (BCP). The lattices were embedded in wax (McMaster-Carr, Robbinsville, NJ) for machining into cylinders 5 mm in diameter and 8 mm in height. The wax was burned out by heating the cylindrical scaffolds to 525  C and holding them at temperature for 1 h. Scaffolds were sterilized by autoclave prior to being implanted. Pore size was determined using two methods. The linear intercept method [39], which is very commonly used for polycrystalline materials to determine grain size, was used to analyze micropore size using backscatter electron mode (BSE) scanning electron microscopy (SEM) images (Phillips XL30 ESEM-FEG, FEI Company,

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Hillsboro, OR). Details are described by Cordell [36]. Briefly, random lines were drawn on the micrographs and the average line length intersecting the pores was reported as the average pore size. Two samples and at least 12 images from each sample were used. The microporosity of the BCP scaffolds was further analyzed via mercury intrusion porosimetry using a Micrometrics Autopore II 9220 (Micrometrics Instrument Corporation, Norcross, GA). 2.2. Surgical procedures Twelve six-month-old, 80 kg male Yorkshire pigs were used for this study. All animal experiments conformed to the University of Illinois Institutional Animal Care and Use Committee (IACUC) guidelines. Three 5 mm diameter bicortical defects were created in the ramus of both hemi-mandibles of each pig using a trephine. The BCP scaffolds were press-fit into the defects. Animals were euthanized at 3, 6, 12, and 24 weeks and scaffolds were recovered and evaluated using histology, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Non-critical sized defects were used here to ensure complete bone regeneration, which is necessary to evaluate bone ingrowth into both macro- and micropores for the first time. Immediately prior to surgery, a sedative cocktail of 1.47 mg telazol, 0.09 mg atropine, 2.9 mg xylazine, and 5.9 mg ketamine per kg body weight was administered. Animals were maintained under general anesthesia with halothane gas in oxygen. Retromandibular and submandibular incisions were made through skin and subcutaneous tissues, preserving the facial artery and vein. Dissection was carried down to the inferior border of the mandible. The periosteum was carefully reflected to expose the mandibular cortex. Three bicortical circular defects measuring 5 mm in diameter were created in the ramus of each hemi-mandible with a trephine bur using a variable speed surgical drill with continuous irrigation. Following the implantations, the periosteum was closed over the bone and sutured with interrupted VicrylÔ suture followed by similar suturing of the muscle layer. Adipose and skin were closed with a continuous VicrylÔ suture. Postoperative analgesics and antibiotics were administered to provide spectrum coverage. Animals were returned to their individual pens to recover. They were maintained on a soft diet for 7–10 days and then resumed a regular dry swine diet until the end of the study. 2.3. Scaffold preparation for histology and scanning electron microscopy To retrieve the scaffolds at the specified survival times, hemi-mandibles were harvested and trimmed using a band saw. The samples were embedded in PMMA according to the methods of Sterchi and Eurell [40]. Briefly, scaffolds and surrounding bone were fixed in 10% neutral buffered formalin, dehydrated in an ethanol series and infiltrated with methylmethacrylate (MMA) monomer. Following polymerization, samples were sectioned parallel to the long axis of the cylinder using a Buehler Isomet 100 diamond saw (Buehler, Lake Bluff, IL) to yield approximately five 500 mm-thick sections, which were affixed to acrylic slides. Sections were polished down to 150 mm thickness, stained with Sanderson’s Rapid Bone Stain (Surgipath, Richmond, IL) and counterstained with acid fuchsin. Control, nonimplanted sections were prepared in the same manner. Sections were imaged using an IX51 light microscope (Olympus, Center Valley, PA). As-fabricated scaffolds and all unstained histology slides were coated with goldpalladium (AuPd) for SEM imaging. Scaffolds and histology slides were imaged in secondary electron (SE) mode using a Hitachi S-4700 SEM (Hitachi High Technologies American, Inc, Pleasanton, CA) and a JEOL 6060 SEM (JEOL Ltd, Tokyo, Japan). Histology slides were also imaged in backscatter electron (BSE) mode using a Phillips XL30 ESEM-FEG (FEI Company, Hillsboro, OR). Energy Dispersive Spectroscopy (EDS) was performed on carbon-coated, acrylic histology slides with a JEOL 7000F SEM (JEOL Ltd).

3. Results 3.1. Scaffold characteristics The scaffolds have a lattice-like architecture consisting of alternating layers of orthogonal rods 394 mm in diameter (Fig. 1a). The rods have a center-to-center, in-plane and out-of-plane spacing of 753 mm and 646 mm, respectively (Fig. 1b, c), where the space between the rods constitutes the macropores. The fugitive PMMA porogen added to the ceramic slurry during scaffold fabrication generates micropores 5.31  4.07 mm in diameter (Fig. 1d), as measured by the line intercept method in a previous study [36]. Analysis using MIP gives an average pore connection size of 2.15 mm and rod microporosity of 46% after sintering (Fig. 2). X-ray diffraction (XRD) analysis shows that the composition of the sintered ceramic is 87% hydroxyapatite and 13% b-tricalcium phosphate (Fig. 3).

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Fig. 1. Macroarchitecture and microstructure of biphasic calcium phosphate scaffolds. a, Biphasic calcium phosphate (BCP) scaffold fabricated by micro-robotic deposition (5 mm ø  8 mm) and implanted into the pig mandible. b, Schematic showing a top view of the scaffold unit cell where the in-plane macropore is indicated by the black arrows. The size of the macropore is 359 mm, rod diameter is 394 mm, and the center-to-center, in-plane spacing is 753 mm. c, Schematic showing a side view of the unit cell. The size of the macropore is 252 mm (orange arrow) and the center-to-center, out-of-plane spacing is 646 mm. d, SEM micrograph of the surface of a microporous rod where micropores are 5.31  4.07 mm.

3.2. Histological evidence of bone, soft tissue, and vessels in macropores Characteristics of normal, functional bone and the associated processes of bone formation and remodeling are apparent in the macropores at all time points, shown using optical histology images (Fig. 4). Mineralized bone in the macropores directly apposes the microporous rods (Figs. 4 and 6a, c, e). Active osteoblasts align along osteoid seams adjacent to mineralized bone (Fig. 4b). Blood vessels, including arteries and veins of varying sizes, are present throughout the macropore space (Fig. 4c). These vessels are essential to the viability of the tissue as bone formation and remodeling are not possible without stable mechanisms of nutrient and waste transport [41,42]. Osteoclasts attach to bone surfaces in

areas of active resorption that are characterized by scalloped edges (Fig. 4d). Finally, adipocytes, which are constituents of healthy bone marrow, reside in the loose connective tissue in the macropores (Fig. 4d). The histology images shown throughout the paper are representative of all time points and many figures include images of samples from multiple time points. As-fabricated, non-implanted (virgin) BCP scaffold sections were prepared for histology as controls in order to determine whether there were any artifacts associated with the stains (Fig. 5). A virgin BCP scaffold stained with Sanderson’s Rapid Bone Stain results in a blue hue (Fig. 5a) where the micropores do not stain. Acid fuchsin does not change the color of the virgin scaffold (Fig. 5b). Sanderson’s Rapid Bone Stain followed by acid fuchsin counterstain, the

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4 6 Pore diameter (µm)

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Fig. 2. Micropore size data. Mercury intrusion porosimetry results show that the average pore interconnection size is 2.15 mm. In addition, micropore interconnectivity is 100% and rod porosity is 46%.

Fig. 3. X-ray diffraction (XRD) spectrum showing phases present. The dominant phase is hydroxyapatite with a minor phase of 13% b-tricalcium phosphate (C). This combination of phases is referred to as biphasic calcium phosphate (BCP). XRD scans were performed at 2Q between 20 and 80 with CuKa radiation at a scan rate of 3 / min and a step size of 0.048 .

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Fig. 4. Natural bone deposition and resorption processes occur within biphasic calcium phosphate scaffold macropores following in vivo implantation. a, Bone (q) deposition occurs in scaffold macropores (*) adjacent to scaffold rods (+). Scaffold rods stain pink non-uniformly. b, Osteoblasts (/) deposit osteoid (8) that subsequently undergoes mineralization. c, Blood vessels of different sizes, including both veins ( ) and arteries (<) are in the macropore space. d, Osteoclasts ( ) resorb bone in macropores which results in scalloped bone edges. Adipocytes constitute some of the loose connective tissue in the macropore space not filled with bone and their presence is associated with bone marrow development (A). Scale bars: 500 mm (a), 100 mm (b–d). Survival times: 24 weeks (a–d). [Mineralized bone ¼ pink/red, Osteoid ¼ blue/purple, Soft tissue ¼ blue, Cell nuclei ¼ dark blue, Cell cytoplasm ¼ light blue.].

combination used for histology of the implanted scaffolds, gives a pink hue to the scaffold (Fig. 5c). These results are in contrast to scaffolds with microscale osteointegration (Figs. 4, 6–9) where micropores are coated or filled with bright and opaque red or blue similar to staining of bone and osteoid, respectively, formed in scaffold macropores. These images were captured at 40 magnification, which is also the magnification of the optical histology images in Figs. 6, 7, 8c, and 9b. 3.3. Histological evidence of bone, osteoid, and cells in micropores Mineralized bone (red), osteoid (dark blue/purple), and cells (dark blue nucleus, light blue cytoplasm) inside of the micropores are shown using complementary pairs of optical and BSE mode scanning electron microscopy (SEM) images (Figs. 6, 9). Red micropores in optical images indicate the presence of mineralized bone (Figs. 6a, c, e, 9b, d). A BSE SEM image (Fig. 6b) shows density contrast between the scaffold (white) and the mineralized bone that fills the micropores (light grey) near the diffuse bone–scaffold interface. The scaffold rod appears brighter in regions corresponding with areas in Fig. 6a in which mineralized bone in the macropore is continuous from the macropore into the micropores. Micropores that appear black or dark grey in BSE SEM images are either empty or contain a cell, soft tissue, osteoid, or partially mineralized bone. For cells, osteoid, or other soft tissue, the density contrast is often not significant enough to clearly

differentiate them from the PMMA embedding medium in the BSE SEM image (see Fig. 9). Some micropores that are not completely filled with mineralized bone stain with a red/pink ring or border on the surface of the micropore (Fig. 7a) and the center of these pores stain blue or purple (Fig. 7a), like osteoid in the macropore space (Fig. 4b). Fig. 7b is a complementary BSE SEM image to Fig. 7a that shows a ring of mineralized bone on the surface of the micropore. Fig. 6c–f shows cells inside micropores. Some of the micropores stain in a manner that is indicative of a pore containing a cell, but no mineralized matrix (Fig. 6c); the pore contains the cell nucleus (dark blue), which is surrounded by cytoplasm (light blue). In other cases, cells are trapped in the mineralized matrix and the staining is similar to that of osteocytes found in bone in the adjacent macropore (Fig. 6c, e). Here, the cell nucleus and cytoplasm are visible, but are surrounded by red-stained mineralized matrix. Cells trapped in mineralized matrix are also visible using BSE SEM (Fig. 6d, f). The individual cells can occupy most of the micropore, or a fraction of the micropore (Fig. 6c, d). In Fig. 6f, the cell inside the micropore has multiple processes extending into the mineralized matrix. There are no visible differences in the morphology of cells embedded in matrix in micropores and the morphology of osteocytes found in bone in the adjacent macropore (Fig. 6f). The images in Fig. 7 suggest that the process of bone formation in micropores begins with mineralization on the HA surface and then fills in toward the center of the micropore. Fig. 7a shows

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micropores that stain with a defined pink ring and a blue/purple interior. BSE SEM of the region highlighted in Fig. 7a confirms the presence of a bone ring coating the surface of the micropores (Fig. 7b). This result from the embedded and polished section is consistent with the features on the surface of a fixed, critical point dried, and then fractured scaffold that had been implanted (Fig. 7c). The HA fracture surface is mostly smooth, but the HA surface open to the micropores in the region shown has a rough, textured coating of uniform thickness. The microstructure of the surface of an asfabricated, non-implanted scaffold rod is shown in Fig. 1d. The interiors of some micropores in Fig. 7 are filled with less dense material that likely consists of cells and proteins, and immature bone at varying stages of development and mineralization. Fig. 7d shows a micropore from a fracture surface that appears to be almost completely filled with bone. SEM and complementary optical images show that osteoclasts not only resorb bone in the micropores, in addition to bone in the macropores, but also degrade the BCP scaffold. Fig. 8a shows scaffold rods with non-uniform staining, indicating non-uniform bone fill in the micropores. The non-uniformity in color in Fig. 8a correlates well with the non-uniform brightness of the rods in the corresponding secondary electron (SE) SEM micrograph (Fig. 8b). Fig. 8c shows the same region at higher magnification. Here, multiple osteoclasts align along surfaces of neighboring rods. Notably, the region directly beneath the osteoclasts is unstained and therefore is not easily distinguished from the adjacent soft tissue in the macropore. A- SE SEM image of the same region (Fig. 8d) clearly shows thinned regions of BCP that correspond to the unstained regions in Fig. 8c. 3.4. Energy dispersive spectroscopy analysis of scaffold rods

Fig. 5. As-fabricated, non-implanted BCP scaffold sections stained to determine whether there were any staining artifacts. a, Sanderson’s Rapid Bone Stain results in a scaffold with a blue hue. The micropores do not stain. b, Acid fuchsin does not change the color of the scaffold. c, Sanderson’s Rapid Bone stain followed by acid fuchsin counterstain gives a pink hue to the scaffold. These results are in contrast to scaffolds with microscale integration (Fig. 4, 6–9) where micropores are coated or filled with bright and opaque pink or blue similar to staining of bone and osteoid, respectively, formed in scaffold macropores. Acid fuchsin stain is water-soluble so the pink hue of the control scaffold could be removed through rinsing with water. These images were captured at 40 magnification, which is the magnification of the optical histology images in Figs. 6, 7, 8c, 9b.

Energy dispersive spectroscopy (EDS) data complement optical histology and BSE SEM images through the detection of calcium and phosphorus. Fig. 9 shows low and high magnification optical and BSE SEM images of a region in a rod analyzed using EDS. Representative EDS spectra are found in Fig. 10. EDS analysis confirms the presence of calcium (Ca) and phosphorus (P) in area 1, a region that is clearly BCP based on examination of the SEM micrograph (Fig. 9c, d). Area 2 is located in the macropore space in bone that apposes the rod while area 3, which is within a micropore that is open to the macropore space, is continuous with area 2. The EDS spectra representing these regions contain Ca and P peaks confirming the presence of mineral thus complementing the histology results. Ca and P are detected in areas 5 and 7, but not area 4. Based on the histology image, area 7 is a micropore that appears to be completely filled with mineralized bone and looks similar to area 3 in the SEM image. Area 5 is also stained pink/red indicating mineralized bone, but looks similar to pores in the SEM image that do not contain Ca and P, like area 4. In the histology image, area 4 is unstained, like BCP (area 1). However, the corresponding BSE SEM image shows that area 4 is a pore, not BCP, and therefore is devoid of partially or fully mineralized bone, osteoid, or cells, and is instead filled with the PMMA embedding medium. Spectra of the pores marked by areas 6 and 8 show the presence of Ca and P, but stain blue/purple which, taken together, indicate the presence of osteoid in the initial stages of mineralization. These examples, particularly areas 4, 5, 6, and 8, show how the use of BSE SEM, EDS and optical imaging are useful in combination to characterize bone within scaffold micropores. 4. Discussion This study demonstrates multiscale osteointegration, which we describe as extensive bone growth into both macro- and micropores. Bone and/or osteoid penetrate microporous BCP rods 394 mm

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Fig. 6. Osteogenic cells initiate bone formation in micropores leading to multiscale osteointegration. a, Micropores stain pink/red indicating the presence of bone. b, The corresponding BSE image shows density contrast between the scaffold (white) and mineralized bone filling the micropores (light grey) from the macropore close to the scaffold rod–bone interface. The regions of the rod in which micropores appear black or dark grey can contain micropores that are partially filled with mineralized bone, cells, unmineralized matrix (i.e. osteoid), or a combination. Some pores may contain only the PMMA embedding medium. c, Micropores occupied by cells that are visible in the optical images are characterized by dark blue nuclei and light blue cytoplasm. The micropores are empty or partially mineralized (<) or in some cases cells occupy a mineralized micropore (/). d, BSE SEM shows cells embedded in the bone matrix that is continuous from the bone occupying the adjacent macropore. e, The morphology of cells in the micropores ( ) is no different from that of the osteocytes found in bone adjacent to the scaffold rod (8). f, BSE SEM shows cell processes extending into surrounding bone matrix. Scale bars: 50 mm (a, c, e). Survival times: 3 weeks (a, b), 12 weeks (c–f). [Mineralized bone ¼ pink/red, Osteoid ¼ blue/purple, Soft tissue ¼ blue, Cell nuclei ¼ dark blue, Cell cytoplasm ¼ light blue.].

in diameter and osteogenic cells reside in the micropores. In Fig. 6, multiple rods appear circular, which is indicative of the histology section being perpendicular to the axis of the scaffold. This allows bone in the center of the rods to be imaged. The exploitation of microporosity as space for bone ingrowth is a novel approach to enhancing osteointegration and the presence of bone within micropores may indicate a degree of osteoinductivity for microporous BCP scaffolds. While this work is the first that specifically focuses on bone growth into micropores in scaffolds with multiscale porosity, we note that the implanted scaffolds also contain all of the elements of fully functional bone within macropores. The macropores are well-vascularized with both veins and arteries of

varying sizes (Fig. 4). Bone is actively remodeled through the normal processes of bone deposition and resorption, as evidenced by scalloped bone edges (Fig. 4d), and osteoclasts actively modify the surface of the microporous rods and the bone contained within the micropores (Figs. 8c, 9b, d). The majority of the work in the literature describing design and evaluation of synthetic bone scaffolds focuses on macroporosity. There have been some important studies that explore microporosity in the struts of CaP-based scaffolds for the purpose of improving osteoconductivity and eliciting osteoinductivity [29,33]. Habibovic et al. reported that microporosity elicits osteoinductive effects in both ectopic sites and critical size bone defects. While

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Fig. 7. Bone formation in biphasic calcium phosphate scaffold rods begins with coating of the micropores. a, Histological staining shows micropores with a defined coating of red/ pink staining indicative of mineralized bone ( ). The interior of the pores are stained blue/purple similar to osteoid or other soft tissue. b, BSE SEM of the same area highlighted in (a) shows the bone coating ( ). c, BSE SEM of a fracture surface from an implanted scaffold shows a uniform coating of the micropores. The coating, w1 mm thick, is textured (8). This is in contrast to the smooth fracture surfaces of BCP (+). The interiors of some micropores appear to be filled with unmineralized tissue (<), consistent with (a) and (b). d, BSE SEM of a fracture surface from an implanted scaffold showing a micropore of the BCP scaffold (+) that is almost completely filled with bone (>). Scale bars: 50 mm (a), 12.5 mm (a, inset). Survival times: 12 weeks (a–c), 6 weeks (d). [Mineralized bone ¼ pink/red, Osteoid ¼ blue/purple, Soft tissue ¼ blue, Cell nuclei ¼ dark blue, Cell cytoplasm ¼ light blue.].

Fig. 8. Osteoclasts on the rod surface resorb bone in the micropores and degrade the biphasic calcium phosphate scaffold rod. a, Optical images show varying amounts of bone fill within the scaffold rods based on the intensity and uniformity of pink stain. b, The areas with the most intense stain in (a), indicating the most bone fill, are also the brightest in the SE SEM image (b). c, Osteoclasts on the surface of the scaffold rods (<). The scaffold directly beneath them is not stained pink. This is indicative of the absence of bone in the micropores. d, The corresponding SE SEM image shows that BCP is still present in the unstained regions beneath the osteoclasts, but that it is noticeably thinned and therefore darker. Scale bars: 200 mm (a) and 50 mm (d). Survival times: 12 weeks (a–d). [Mineralized bone ¼ pink/red, Osteoid ¼ blue/purple, Soft tissue ¼ blue, Cell nuclei ¼ dark blue, Cell cytoplasm ¼ light blue.].

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Fig. 9. EDS analysis confirms presence of calcium and phosphorus in micropores that stain pink. a, b, Low magnification BSE SEM and optical images indicate the region in which EDS was performed. The arrow in (b) shows an osteoclast. Note the region near the surface of the scaffold rod beneath the osteoclast is unstained, but is clearly biphasic calcium phosphate (BCP), as shown in (a). c, d, EDS analysis confirms that area 1, a region of BCP, contains calcium (Ca) and phosphorus (P). Areas 2, 3, 5, and 7 contain Ca and P and are stained red/pink indicating the presence of mineralized bone within micropores. Areas 6 and 8 contain Ca and P and are stained blue/purple, which may be due to the presence of osteoid undergoing the initial stages of mineralization. Area 4 does not contain Ca or P. It is not stained pink in (d) and (c) shows that it is a micropore so it is a micropore filled only with PMMA. Scale bars: 50 mm (a) and 15 mm (d). Survival times: 12 weeks (a–d). [Mineralized bone ¼ pink/red, Osteoid ¼ blue/purple, Soft tissue ¼ blue, Cell nuclei ¼ dark blue, Cell cytoplasm ¼ light blue.].

they reported improved bone formation in macropores, there was no report of growth into micropores [33]. Hing et al. also demonstrated accelerated bone growth and more complete bone regeneration within macropores of CaP scaffolds with microporous scaffolds struts [29]. In a different study they showed viable cells and bone in macropores less than 100 mm in size but, again, did not show bone in micropores less than 10 mm [43]. Studies by our group and others suggested that bone forms in micropores at the surface of rods [27,28,30,35]. However, the evidence was not definitive. Porter et al. have shown evidence of bone infiltrating micropores that are open to macropores on the surface of CaP scaffold struts; mineralized collagen fibrils penetrated micropores at the surface of a CaP strut, but this was limited to the scaffold–bone interface [34]. Previous works highlight one dimension of the importance of scaffold microporosity such as increased bone regeneration and osteoconductivity as well as osteoinductive potential associated with bone formation in macropores [27–29,31,33]. Our results provide another important dimension: the inclusion of microporosity in these macroporous scaffolds can result in multiscale osteointegration and thus a living composite material. There is no ‘‘dead space’’ in rods or struts in these scaffolds that is present in CaP scaffolds with only macroscale osteointegration. Bone formation throughout the BCP scaffold rods in this study can likely be attributed to the presence of osteogenic cells within micropores. The migration of osteoprogenitor cells into micropores and their subsequent differentiation followed by formation of osteoid and mineralized tissue in microporous rods occur without any ex vivo surface modification or the addition of growth factors or cells. Such cell migration through the micropores and the subsequent bone formation may be a novel indicator of scaffold osteoinductivity. The synergistic combination of the material

properties, the macroarchitecture (rod spacing, diameter), and the interconnected microporosity together are likely to facilitate this process. Any cells that may be present in the microporous rods are likely to retain access to nutrients and expel waste by the same mechanism as osteocytes do in cancellous bone, through the lacunar–canalicular network. In vivo, cells like osteocytes embedded in mineralized matrix in trabeculae, must reside within 200 mm of a vascular supply to remain viable [44]. Cells at the center of the 394 mm diameter rods meet this requirement since the maximum distance between the cells at the center and the macropore space is less than 200 mm. Previous work investigating the osteoinductivity of certain scaffolds suggests that dissolution/re-precipitation of CaP leads to the formation of a biological apatite layer on the scaffold surface [31,45]. For the scaffolds studied here, such modifications could take place because the BCP scaffold possesses interconnected micropores and the more soluble b-TCP phase provides a microenvironment conducive to dissolution/re-precipitation, which can then lead to the formation of a biological apatite layer. Cells may recognize this apatite layer based on physicochemical features and such features may drive migration, differentiation, and bone formation within micropores [46,47]. While biological apatite may form in these scaffolds to some degree, we assert that the material present on the micropore walls that we have identified as bone matrix at various degrees of mineralization cannot be biological apatite. The red staining of mineralized bone by acid fuchsin (AF) results from interactions between AF and the collagen in the bone matrix. AF is an anionic molecule under acidic conditions, due to the presence of sulfonic groups [48], and is often utilized as a stain for collagen such as in the case of van Gieson’s picrofuchsin [49]. The mechanism of

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Fig. 10. EDS spectra corresponding to the regions shown in Fig. 4. Area 1, a region of BCP, contains calcium (Ca) and phosphorus (P). Ca and P signals are also detected in area 2, which includes bone in a macropore adjacent to the scaffold. The EDS spectrum confirms that area 3, a region of bone continuous with area 2, is mineralized. Area 4 is an empty pore based on histological staining and BSE SEM imaging and EDS evaluation did not result in detection of Ca or P signals. Areas 5–8 all produce some Ca and P signals and contain bone based on histological staining evidence.

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staining has not been fully elucidated, but does include electrostatic, and likely hydrophobic, interactions. The general consensus in the histology community is that the anionic groups of AF neutralize positively charged amino acid residues in collagen molecules [50,51]. Therefore, in order for the AF to stain biological apatite, either the apatite must contain collagen or the AF would have to stain the non-collagenous proteins in the apatite. Neither is the case and in addition, there is no evidence or suggestion in the histology literature that AF stains biological apatite. The exact protein composition of biological apatite is not well characterized, but it does include bone-associated non-collagenous proteins [4,14,31,45]. The vast majority of the non-collagenous bone proteins, such as osteocalcin, which are thought to play an important role in bone matrix mineralization, are highly acidic [52–54]. These proteins generally contain a long, negatively charged sequence that, coupled with the protein conformation, allow for formation of coordination bonds with Ca2þ ions in calcium phosphate [52]. AF, being negatively charged under acidic conditions, is very unlikely to interact with the acidic proteins, and collagen, which is known to bind AF, is not present in biological apatite. Osteoid stains blue or blue/purple with Sanderson’s Rapid Bone Stain (SRBS) and AF counterstain, rather than red, even though it contains mostly collagen. This is because of the difference in both composition and organization of the collagen in osteoid as compared to mature collagen in bone matrix. Newly formed osteoid has a significant fraction of mucopolysaccharides along with low density and less organized collagen fibrils [55]. The mucopolysaccharides are anionic at low pH (negatively charged), which would prevent electrostatic interactions with AF but would promote staining with SRBS. The density, maturity and organization of collagen vary from the surface of the osteoid to the mineralization front, as observed through transmission electron microscopy [55,56]. The collagen matrix has higher fibril density with a more organized structure near the mineralization front [55,57,58]. Flint et al. showed that AF has stronger interactions, or is better retained, in more highly ordered collagen such as that found in the tensioned collagen of tendon versus dermal collagen [59]. Similarly, dermal collagen that has been stretched retains AF better as compared to unstretched tissue [59]. MacConaill referred to acceptance of AF as erythrophilia and reported that collagen erythrophilia increases with collagen maturity [60]. Some interaction between collagen and AF in osteoid likely adds a component of red staining to the primary blue staining from SRBS, which results in an overall purple color for the work shown here (e.g. Fig. 4b). Figs. 6, 7 and 9 showed histology images and corresponding BSE SEM images. At the bone/scaffold rod interface, the bone in the micropores had the same density contrast as the bone in the macropore space. However, for other regions in the rod, the density of the material contained in some of the micropores is more similar to the PMMA embedding medium, even though the corresponding histology image shows bone in the micropore. This discrepancy is related to the maturation of the bone in the micropore and emphasizes the importance of using both BSE SEM and histology images for characterization. While osteoid mineralization can begin in as few as 5–10 days coinciding with the final stages of collagen maturation and organization, complete mineralization can take as long as 3–6 months [61,62]. During this extended time period the density of the osteoid/maturing bone increases as mineral crystals grow [63]. Because the matrix is primarily high density, ordered, mature collagen when mineralization begins, the collagen stains red by AF. However, the density of the mineral may continue to increase. This explains why the bone in the micropores stains red in locations where the BSE SEM shows less dense material in the pores compared to the density of bone in the macropore space between scaffold rods.

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Ultimately, the objective in studying these BCP scaffolds with macro- and microporosity is to address the persisting need for scaffolds that fulfill transport and mechanical needs both in the short and the long term. These scaffolds have shown potential for load-bearing applications in other studies [24–27] and the relatively slow degradation of BCP provides initial mechanical stability that persists in the long term. The multiscale osteointegration shown here has important implications for improved overall performance of scaffold/bone composites. Extensive bone growth into micropores increases the bone/scaffold interface area by orders of magnitude over scaffolds without microporosity. The increase in surface area means more effective and more efficient load transfer between bone and scaffold by two mechanisms. First, the inherent bioactivity of CaPs allows for a chemical bond between the bone and scaffold and, second, the multiscale osteointegration provides mechanical interlock of the two phases [64–66]. Additionally, the bone-filled microporous rods yield a composite with no ‘‘dead space’’. This results in potential for a significantly tougher composite compared to a scaffold without bone-filled micropores or with no micropores at all. The filled pores provide a mechanism to deflect, blunt, or slow crack growth in the scaffold. A final implication of this living composite is that cells embedded in bone in micropores are analogous to osteocytes in lacunae in the adjacent macropore bone. The presence of osteocytes in micropores throughout the rods provides a unique opportunity for a continuous mechanosensory network unlike any other scaffold system shown to date. 5. Conclusions In this paper we exploit microporosity specifically for the purpose of multiscale osteointegration in CaP-based scaffolds for bone tissue engineering and demonstrate multiscale osteointegration in these scaffolds. Our approach contrasts the more standard approach of optimizing scaffold macroarchitecture. The unique combination of open macropores and interconnected micropores within scaffold rods encourages osteoprogenitor and/or osteogenic cells to populate the 394 mm diameter rods, resulting in osteoid and mineralized bone coating the walls of the rod micropores. The exploitation of microporosity to achieve multiscale osteointegration offers a new paradigm for scaffold design and may lead to successful development and translation of these BCP bone scaffolds to load-bearing applications. Acknowledgements This work was supported by grants from the Aircast Foundation (no. S0406R) and by a Research Support Grant award from the Oral and Maxillofacial Surgery Foundation. This work was carried out in part in the Frederick Seitz Materials Research Laboratory Central Facilities, University of Illinois, which are partially supported by the U.S. Department of Energy under grants DE-FG02-07ER46453 and DE-FG02-07ER46471. The authors thank J. Cordell, D. Hoelzle, C.J.Park, M. Poellmann, and S. Wilson for assistance with all surgical preparation and procedures and specimen recovery, M. Vogle for assistance with histology, and S. Robinson, J. Mabon, V. Petrova, and C. Wallace for assistance with scanning electron microscopy imaging and energy dispersive spectroscopy data collection. Fellowship assistance was awarded to SKLL by the National Science Foundation Graduate Research Fellowship program, the National Defense Science and Engineering Graduate Research Fellowship program, and the University of Illinois at Urbana-Champaign (UIUC) Support for Under-Represented Groups in Engineering (SURGE) Fellowship Program. Fellowship assistance was awarded to SJP by the UIUC College of Engineering Roy J. Carver Fellowship program and the UIUC SURGE Fellowship Program.

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