hydroxyapatite-based multilayer composite scaffold for hard tissue repair

Accepted Manuscript Decellularized bovine small intestinal submucosa-PCL/ hydroxyapatite-based multilayer composite scaffold for hard tissue repair

Mahmut Parmaksiz, Ayşe Eser Elçin, Yaşar Murat Elçin PII: DOI: Reference:

S0928-4931(18)30844-0 doi:10.1016/j.msec.2018.10.011 MSC 8937

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

21 March 2018 14 September 2018 2 October 2018

Please cite this article as: Mahmut Parmaksiz, Ayşe Eser Elçin, Yaşar Murat Elçin , Decellularized bovine small intestinal submucosa-PCL/hydroxyapatite-based multilayer composite scaffold for hard tissue repair. Msc (2018), doi:10.1016/j.msec.2018.10.011

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ACCEPTED MANUSCRIPT Decellularized bovine small intestinal submucosaPCL/hydroxyapatite-based multilayer composite scaffold for hard tissue repair Mahmut Parmaksiz a, Ayşe Eser Elçin a, Yaşar Murat Elçin a,b* Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University

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a

Biovalda Health Technologies, Inc., Ankara, Turkey

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b

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Corresponding author: Professor Dr. Y. Murat Elçin

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*

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Faculty of Science, and Ankara University Stem Cell Institute, Ankara, Turkey

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Mailing Address: Ankara University, Faculty of Science, Biochemistry Division, Tandogan, 06100 Ankara, Turkey.

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Tel: +90-(312)-212 6720, Ext.1096. Fax: +90-(312)-223 2395

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E-mail-1: [email protected] (Y.M. Elçin); E-mail-2: [email protected] (Y.M. Elçin)

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Running Title:

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Decellularized bSIS-PCL/HAp composite scaffold

Author disclosure statement YME is the founder and shareholder of Biovalda Health Technologies Inc. (Ankara, Turkey). The authors declare no competing financial interests in relation to this article. The authors are alone responsible for the content and writing of the paper.

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ACCEPTED MANUSCRIPT Decellularized bovine small intestinal submucosaPCL/hydroxyapatite-based multilayer composite scaffold for hard tissue repair

ABSTRACT

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This study involved the development of a multilayer osteogenic tissue scaffold by assembling decellularized bovine small intestinal submucosa (bSIS) layers, together with synthetic

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hydroxyapatite microparticles (HAp) and poly(ε-caprolactone) (PCL) as the binder. As a first

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step, the surface and mechanical properties of the developed scaffold was determined, after which the biocompatibility was evaluated through seeding with isolated rat bone marrow

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mesenchymal stem cells (BM-MSCs). Then, a 21-day culture study was performed to investigate the in vitro osteoinductive potential of the scaffold on BM-MSCs under standard

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and osteogenic culture conditions. The SEM findings indicated that a uniform multilayer and perforated structure was acquired; that the HAp microparticles were homogenously

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distributed within the structure; and that the PCL-bound laminar scaffold had structural

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integrity. Mechanical tests revealed that the scaffold maintained its mechanical stability for at least 21 days in culture, with no changes in the first-day maximum strength and maximum stress values of 625.123 ± 70.531 N and 6.57762 ± 0.742 MPa, respectively. MTT and SEM

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analyses together revealed that BM-MSCs preserved their viability and proliferated during a 14-day culture period on the multilayer scaffold. Immunofluorescence analyses indicated that

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cells on the scaffold differentiated into the osteogenic lineage, by the culture-time-dependent increase in osteogenic markers’ expression, i.e. Alkaline phosphatase, Osteopontin, and Osteocalcin. It was also clear that, the osteoinductive effect by the composite scaffold on BMMSCs could be achieved even without the use of any external osteogenic inducers.

Keywords: Osteogenic scaffold; decellularized SIS; hydroxyapatite; multilayer hybrid template; bone tissue engineering 2

ACCEPTED MANUSCRIPT 1. Introduction Bone tissue engineering aims to develop osteogenic scaffolds with the ability to induce bone regeneration and accelerate integration with the adjacent tissue [1]. The major parameters to be considered during development of an osteogenic scaffold biomaterial include the availability of cell attachment sites, adequate biomechanical properties, ability to be

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involved in the bone remodeling process, as well as a pore size of ~100-300 µm, porosity

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level usually >70%, and a pore connectivity suitable for cell migration and proliferation [2,3].

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To ensure the biomimetic/osteogenic and load-bearing properties, this biomaterial should preferably incorporate bone proteins and osteoconductive molecules [4].

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At the present time, osteogenic scaffolds are commonly based on ceramics, polymers (natural or synthetic), or their composites. An examination of these biomaterials reveals a

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predominant use of bioceramics based on calcium phosphates, the major inorganic component of natural bone [5-11]. In particular, hydroxyapatite (HAp) has widespread use in the clinics

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as an orthopedic/dental bioceramic material [12,13]. HAp is osteoconductive; its

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biocompatibility and inflammatory properties can be adjusted according to its shape and size [14]. Notwithstanding these predominant properties, the use of HAp as a stand-alone load-

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bearing scaffold is limited by its insufficiency in the post-application re-modelling process, as well as the production challenges associated with its processing that stem from its mechanical

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properties, i.e. toughness and lack of elasticity [14,15]. On the other hand, natural polymers such as collagen, chitosan and silk are relatively easy to process in the production of bioscaffolds, and resemble more closely to the organic component of the natural bone matrix [16-18]. Biocompatible synthetic polymers such as poly(ε -caprolactone) (PCL), poly (D,Llactic acid) (PLA), and poly(lactic-co-glycolic acid) (PLGA) offer certain advantages owing to their ease of processing and their chemical composition that can be adjusted to specific purposes of use [9,10,19-21]. However, they also suffer from disadvantages such as low 3

ACCEPTED MANUSCRIPT mechanical resistance and, in particular, the undesirable effects associated with the local accumulation of the acidic products that results from the rapid degradation of synthetic polymers such as PLA [4,11,20,22]. The approach of preparing composites constituted of polymers and calcium phosphate minerals combines the advantages of these two classes of materials. The composite form

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allows the development of osteoconductive biomaterials that have the appropriate mechanical

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resistance and could resemble natural bone tissue. Composite scaffolds containing collagen,

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the major organic component in natural bone, and HAp have been evaluated by several groups [4,23-25]. It is also known that despite their advantages, these composites are inadequate in

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terms of bioactive components, and fail to fully meet the desired mechanical properties [1,3]. To overcome this issue, active components in the natural bone matrix have been integrated

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into synthetic materials, with a focus on controlling cell behavior in the biomaterial-cell interface [1,26,27]. However, better imitating the rich bioactive content of the natural bone is

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still an endeavor to scientists.

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A current approach which stands out is the use of decellularized ECMs of natural tissues, as a scaffolding material [28]. In this context, the small intestinal submucosa layer

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(SIS) has gained interest in recent years for regenerative medicinal applications. SIS-ECM naturally contains the collagen types and cytokines with critical functions in tissue repair and

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remodeling. Decellularized SIS-ECM from porcine has received FDA approval for a number of clinical applications, including venous ulcers, diabetic ulcers, skin injuries, vascular repair, bladder wall repair, hernia repair, and cartilage regeneration [28-31]. On the other hand, studies in which SIS has been used in bone tissue engineering are very limited. Mesenchymal stem cells (MSCs) have drawn significant attention as a potential cell source for orthopaedic tissue engineering applications [32-34]. MSCs have high proliferation ability, and can be differentiated into the osteogenic lineages. In particular, bone marrow 4

ACCEPTED MANUSCRIPT (BM) derived MSCs can form bone-like tissue once an appropriate osteogenic scaffold and in vitro culture conditions are provided [3,33-36]. In this study, a novel decellularized bovine SIS-ECM, PCL and HAp-based composite multi-layer scaffold was developed for bone tissue engineering applications. First, the surface, mechanical and cell interaction properties of the SIS-PCL/HAp scaffold were investigated,

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after which the in vitro osteoinductive effect of the scaffold was examined through BM-MSC

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cultures. The findings indicate that bovine SIS-PCL/HAp scaffold has the potential to be used

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as a bone tissue scaffold.

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2. Materials and methods

2.1. Materials

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Bovine small intestines (from 20-24 months old male animals; 225±15 kg) were

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obtained from a veterinary-controlled slaughterhouse (Misirdali Et Kombinasi) and were brought to the laboratory in cold phosphate-buffered saline (PBS; pH 7.4) within 4 h after

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sacrifice. Poly(ɛ-caprolactone) having a Mw of 80,000 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxyapatite microparticles (30 µm-size) in PBS was obtained from

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Plasma Biotal (Buxton, UK). All decellularization reagents, buffer solutions, and organic solvents were of analytical grade, and were obtained from Sigma. Cell culture media, supplements and fetal calf serum (FCS) all were purchased from Lonza (Basel, Switzerland). Osteogenic inducers were cell culture grade and were obtained from Sigma. Flow cytometry buffers and consumables were purchased from Becton Dickinson Biosciences (BD; San Jose, CA, USA). IHC antibodies were procured from either Chemicon (Temecula, CA, USA) or from BD. 5

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2.2. Preparation of the bovine SIS-PCL/HAp construct Bovine SIS (bSIS) layers were isolated and decellularized according to the standard protocol optimized for the bovine SIS tissue [29]. This method yields an ECM rich in bioactive content, devoid of any cell remnants. The success of decellularization was

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confirmed by the complete (100%) removal of the cells, reduction of the DNA content to

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˂10% of the native tissue, and preservation of the sulphated glycosaminoglycans (˃5µg/mg

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tissue) [29].

Decellularized bSIS membranes were cut in the form of discs of ~5 mm-diameter to

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prepare the multi-layer composites. Then, fifty bSIS membrane discs were superposed on top of each other by using a least amount of (4 spots ×10 µL per layer) biodegradable glue (10%

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PCL prepared in dichloromethane:methanol (3:1, v:v)) for stabilization of the discs. By using fifty layers of the SIS membranes, it was possible to achieve a construct with a height of ~8

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mm. Then, 5-6 holes (each ~0.5 mm) were drilled through the final laminated cylindrical

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construct (h=~7 mm) using a hand drill from the top. The bSIS-PCL/HAp constructs were sterilized in 70% ethanol for 2 hours and then excessively washed (10 ×10 mL) with sterile

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PBS. The soaked buffer in the constructs were removed by sterile filter paper. Then, a UVsterilized (254 nm; 30 min) suspension of HAp microparticles in PBS was dropwise

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incorporated into the constructs. The amount of incorporated microparticles was adjusted to the dry weight ratio of 1:10 (HAp: construct; i.e. 0.1 mg HAp per scaffold) after optimization of the procedure. The amount of HAp microparticles which fell off the constructs was negligible.

2.3. Cell culture and seeding on constructs

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ACCEPTED MANUSCRIPT Rat bone marrow mesenchymal stem cells (BM-MSCs) were isolated and cultured up to passages 2-4 in the standard culture medium [SM; i.e. DMEM (Dulbecco’s Modified Eagle Medium) supplemented with 10% FCS, 100U/mL penicilin, 100 μg/mL streptomycin], in an incubator at 37°C, 5% CO₂ and 95% humidity by following established method [36]. Medium was replaced every 2 days. Then cells were characterized via flow cytometry. Later, the cells

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were seeded on the scaffolds at a density of ~4.0 × 10⁶ cells/construct, and were cultured upto

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were assessed using the MTT assay at days 1, 3, 7 and 14.

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14 days on multilayer constructs. During the culture period, cell viability and proliferation

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2.4. Osteogenic induction procedure

Following the cell seeding on constructs, osteogenic induction studies were performed

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on two groups: In Group I, at the tenth day of cell seeding, SM was exchanged with the osteogenic induction medium (OM; i.e. DMEM-Low-glucose supplemented with 10-8 M

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dexamethasone, 10 mM Na-β-glycerophosphate and 50mg/mL ascorbic acid), whereas in

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Group II cells seeded constructs were kept in the SM. In both of the groups, cultures were

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maintained for 7, 14 and 21 days.

2.5. Mechanical evaluation of the scaffolds

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To evaluate the mechanical properties of the multilayer constructs, compression test was applied on four types of samples using the universal testing machine (Shimadzu AGS-X, Tokyo, Japan). Samples consisted of cell-free HAp-free multilayer bSIS-PCL (I), cell-free multilayer bSIS-PCL/HAp (II), BM-MSCs-laden multilayer bSIS-PCL/HAp after 21 days of osteogenic induction culture (III) and BM-MSCs-laden multilayer bSIS-PCL/HAp after 21 days of standard culture (IV). Briefly, the constructs were placed at the center of the bottom

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ACCEPTED MANUSCRIPT plate and then the pressure was applied at room temperature, with the operation conditions of 1 mm/min crosshead speed, and 10 mm gauge length in 500 N load capacity.

2.6. Immunohistochemistry and laser scanning confocal microscopy For further analysis, in vitro osteogenic differentiation of BM-MSCs on multilayer

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scaffolds was assessed by using immunofluorescence (IF) markers. Following osteogenic

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induction, cell-seeded constructs were collected from each group at 0, 7, 14 and 21 days; then

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fixed with 10% formaldehyde in potassium-phosphate buffer. Later, the scaffolds were embedded in Jung Tissue Freezing Medium (Leica, Wetzlar, Germany) and sectioned (~8-10

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µm-thick) on microscopic glass slides. The avidin-biotin-peroxidase complex method was utilized for IF analyses of osteogenic differentiation with bone-related IHC markers, i.e.

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alkaline phosphatase (ALP; Santa Cruz Biotechnology Inc., sc-98652), Osteopontin (OPN; Santa Cruz, sc-21742), and Osteocalcin (OSC; Santa Cruz, sc-30044). Collagen I (Col I;

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Santa Cruz, sc- 59772) analysis was cancelled since this marker did not give reliable results in

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preliminary experiments, due to its high availability in decellularized SIS.

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2.7. Scanning electron microscopy (SEM) analysis Following the osteogenic induction, samples from both groups were fixed for SEM to

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evaluate the multilayer composite structure and cell proliferation in terms of substrate coverage. SEM examinations were performed at days 0, 7, 14 and 21. Cell-free multilayer scaffold was taken as a control. At predetermined time points, cell-free and cell-laden scaffolds were fixed with 2.5% glutaraldehyde in cacodylate buffer. Samples were dehydrated in ethanol series (from 60% to 95%), sputter coated with gold and analyzed using a JEOL JSM 5600 model scanning electron microscope (Tokyo, Japan).

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ACCEPTED MANUSCRIPT 2.8. Statistics Unless otherwise noted, all experiments were performed in triplicates. The data were presented as mean±standard deviation. Statistical analyses were conducted using the unpaired Student’s t-test, and a p < 0.05 value was considered to be statistically significant.

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3. Results and discussion

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This study is based on the development of a multilayer composite tissue engineering

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scaffold composed of bioactive decellularized bovine SIS-ECM, biodegradable polymer poly(ε-caprolactone), and hydroxyapatite bioceramic microparticles. After the morphological

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evaluation of the hybrid construct, the scaffolds were seeded with rat BM-MSCs for evaluation of the in vitro cellular interactions, i.e. cell adhesion, proliferation, and osteogenic

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differentiation.

Bone tissue is a natural composite, comprised of the inorganic part, predominantly of

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hydroxyapatite, and ~35% collagen-containing organic fraction [25]. Aside from this

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chemical composition, bone has a complex extracellular matrix which cannot be synthetically imitated [1]. In bone tissue engineering scaffolds–in which composite forms made solely of

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synthetic and/or natural polymers or in combination with HAp– it is considered a limitation that natural bone tissue cannot be successfully emulated due to the insufficient bioactive

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contents of the scaffolds [37,38]. Taking this into consideration, we have adopted in this study a decellularized tissue ECM-based biomaterial approach in which the bioactive content of the natural tissue is largely preserved. In this context, decellularized bovine SIS layers were used to form the composite osteogenic scaffold. Fig. 1 illustrates in a diagram the stages of multilayer bSIS-PCL/HAp construct preparation. Taking into consideration the suitability for use in multi-well cell culture plates, circular SIS-ECM membranes measuring 8 mm in diameter were stacked in 50 layers using adhesive PCL microdroplets to create a cylindrical 9

ACCEPTED MANUSCRIPT form ~5 mm in height (Fig. 1a,b). While bone tissue engineering studies employing a number of multilayer scaffolds have been reported in literature [39,40], this study represents the first to evaluate the combination of bSIS-PCL/HAp. An increase in the synthesis of bone-specific molecules (such as, collagen and osteocalcin) is reported in studies where decellularized SIS is used in powder form within the

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scaffolds. However, these studies also describe limitations concerning mechanical properties

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[8,41]. In the present study SIS-ECM layers were stacked on each other using a midget

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amount of PCL solutions, with the objective to achieve an improvement in the mechanical properties of the final construct, and ensuring that its layers are holding together. Afterwards,

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the multilayer bSIS-ECM/PCL constructs were perforated to support the easy penetration of cells, medium transfer and for supporting anastomosis via new capillary formation within the

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construct in prospective in vivo studies (Fig. 1c). Thus, it is well known that the scaffold porosity in different scales is important both during in vitro cell cultures and also following in

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vivo transplantation [42-44].

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In the final stage of fabrication, the PCL-stabilized multi-layered bSIS-ECM scaffold was composited with hydroxyapatite, ensuring uniformly distribution of ~30 µm HAp

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microparticles in the porous laminar bSIS-ECM structure (Fig. 1d). A large number of studies have been reported in literature in which HAp is used as a component of scaffolds owing to its

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osteoinductive and osteoconductive properties [45,46]. The effect of the scaffold composition on post-implantation inflammatory response is well-known, and even the smallest incompatible component could lead to fibrous encapsulation and insufficient integration. In this study, the size (~30 μm) and form (spherical, with a smooth surface) of the HAp microparticles were determined by taking this into account. Thus, there are studies showing that smaller-sized and needle-like HAp microparticles activate an inflammatory response, accompanied by an increase in the secretion of NLRP3 inflammasome and IL-1β [47]. 10

ACCEPTED MANUSCRIPT The pore structure and the distribution of HAp microparticles within the multi-layered bSIS-PCL/HAp construct were analyzed by SEM. A close examination of Fig. 2 reveals that the bSIS-ECM layers are stacked on top of each other, that there are regularly spaced gaps between the layers (Fig. 2a,c), and that HAp microparticles are homogenously distributed throughout the entire multi-layer structure (Fig. 2b,c). It can also be noted that PCL does not

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spread along each SIS layer while holding the layers together and maintaining the interfacial

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spaces between layers (Fig. 2c). The SEM images show also that HAp microparticles are

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present even in the sectioned regions, and in the parts where holes are drilled (indicated with arrows in Fig. 2b) revealing the relationship between HAp and the inherently present

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collagen/integrins in the bSIS-ECM. In fact, it is known that ECM-integrin ligands induce adhesive formations and activate the cell regulatory signaling pathways. The large surface

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area of the construct, depending on the micro- and macro-porous structure has the potential to accelerate the bone regeneration process during in vivo applications.

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Primary BM-MSCs were used in the in vitro cell interaction studies of SIS-PCL/HAp

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scaffolds. Immunophenotypical analysis of mesenchymal stem cells showed that they express high levels of CD29, CD54, CD73, CD90 and CD105, but do not express CD31, CD34 or

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CD45 (Fig. 3). These findings demonstrate the mesenchymal stem cell character of the cells, which were initially expanded in adherent culture [48].

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The viability of the BM-MSCs seeded on multilayer SIS-PCL/HAp scaffolds was pursued, in terms of cellular mitochondrial dehydrogenase activity by using the MTT assay. It was shown qualitatively (Fig. 4a) and quantitatively (Fig. 4b) that the cells sustained their metabolic activity for a duration of 14 days in culture on the composite scaffolds, with a timedependent increase in proliferation. The images from different time points of the BM-MSC cultures on the SIS-PCL/HAp scaffold indicate that the color (purple) intensity of formazan,

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ACCEPTED MANUSCRIPT the product of the MTT assay, increases on the composite scaffold depending over time (Fig. 4a). It is clear from Fig. 4b that BM-MSCs proliferate on the SIS-PCL/HAp scaffold in a timedependent manner. On the other hand, the proliferation of the cells on the scaffold appears to take place at a relatively lower level than that of the cells grown on a standard culture surface

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(Fig. 4b). However, it is likely that this may have resulted from the technical difficulty in

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completely extracting the formazan crystals, from the porous and mineral-accumulating three-

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dimensional SIS-PCL/HAp construct. Similar findings with other porous scaffolds have been previously reported [10].

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The interaction of BM-MSCs with the bSIS-PCL/HAp scaffold has also been analyzed using SEM. From Fig. 5, it is apparent that the mesenchymal stem cells adhere to both bSIS

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layers and to HAp microparticles via their pseudopods, and the cells start to cover the layers of the composite structure in culture. Fig. 5a shows that bSIS layers are hold together in

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certain order after 14 days of culture. HAp microparticles are visible throughout the construct,

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and even at the perforation regions (Fig. 5b-d). While some of the cells have spread on the layers of the construct (Fig. 5e), some others have positioned in between the neighboring bSIS

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layers through their cellular extensions and ECM (Fig. 5f), demonstrating tissue-like formations. Both the MTT and SEM findings indicate that bSIS-PCL/HAp scaffold is

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compatible to MSCs. This can be explained by the interaction of collagen fibers and sulphated glycosaminoglycans present in the bSIS-ECM, hydroxyapatite, which can form bonds with the polar and polarizable molecules [49-51], as well as specific proteins and polynucleotides contained in the cell culture medium with the cell surface proteins to promote cell adhesion. Another limitation in bone tissue engineering scaffolds is the inadequacy of mechanical properties [11,52]. Scaffolds made up of stand-alone biodegradable synthetic polymers usually fail to reach the targeted mechanical stability and compressive strength 12

ACCEPTED MANUSCRIPT levels, whereas bioceramic scaffolds with a higher compressive strength have disadvantages during the biomaterial fabrication process, and are also incompetent in the re-modelling processes. The use of composite hybrid structures is usually the adopted approach to overcome these limitations [46,53,54]. Findings related to the mechanical characterization of SIS-PCL-HAp composite scaffolds are presented in Fig. 6. At first, the mechanical properties

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of the lyophilized and the wet forms of cell-free bSIS-PCL/HAp were compared. As can be

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seen from Fig. 6a, while the lyophilized form of the cell-free bSIS-PCL/HAp composite

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scaffold exhibited a maximum strength of 2183.98±554.83 N and a maximum stress of 23.4048±5.945 MPa, the measured values for the wet scaffold were 625.123±70.531 N and

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6.57762±0.742 MPa, respectively. Secondly, the mechanical stability of cell-laden constructs were compared under the expansion culture (SM) and osteogenic culture (OM) conditions

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(Fig. 6b). The standard culture conditions (expansion medium) did not have a negative effect on the mechanical properties of the BM-MSC-seeded bSIS-PCL/HAp. Thus, a maximum

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strength of 700.298±48.152 N and a maximum stress of 8.91647±0.613 MPa were determined

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for the cell-laden scaffold after 21 days of culture, which were very close to the values of the cell-free wet bSIS-PCL/HAp construct. On the other hand, a ~25 % decrease in both the

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maximum strength and stress values was measured for the cell-laden scaffolds cultured for 21 days under the osteogenic differentiation conditions (Fig. 6b). It is known that in vitro cell

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cultures usually have a stationary, glycolysis-dependent metabolism in their standard expansion cultures. However, during differentiation the cell metabolism speeds up which leads to the upregulation of oxidative phosphorylation [55]. The change in mechanical properties can be attributed to the osteogenic differentiation processes accelerating the in vitro modeling of cell-laden composite scaffold. It is worth noting that, although the experiments are performed under the in vitro conditions, the compressive force values measured both for

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ACCEPTED MANUSCRIPT the cell-laden and cell-free bSIS-PCL/HAp scaffolds are within the mechanical value range (2-45 MPa compressive force) of the human cancellous bone [56]. The in vitro osteogenic differentiation tendency of BM-MSCs on the bSIS-PCL/HAp scaffold was evaluated by immunofluorescence microscopy (Figs. 7-9). Within this context, cultures of BM-MSC-laden scaffolds which were either kept in standard expansion medium,

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or in osteogenic induction medium conditions [9,10] were compared. The group under SM

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conditions was particularly noteworthy, in that it allows direct evaluation of the potential

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osteoinductive efficiency of bSIS-PCL/HAp on BM-MSCs without the use of any osteogenic differentiation agents.

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Alkaline phosphatase, the first of the designated markers for IF analysis, is a widely used metalloenzyme in demonstrations of the osteogenic activity of bone tissue. Contribution

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of ALP is attributed to its association with the increase in the local concentration of inorganic phosphate [57,58]. While BM-MSCs express a somewhat low level of ALP, this is not intense

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enough to overshadow the dramatic increase in the ALP expression during osteogenic

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differentiation. IF micrographs of ALP stained samples from the 7-, 14- and 21-day cultures are collectively presented in Fig. 7. IF findings point to a gradual, time-dependent increase in

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the ALP expression of both of the groups, compared to that of the starting (Day 0) culture. Furthermore, it is observed that the ALP expression in the OM group under the influence of

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osteoinductive medium conditions is slightly higher when compared to the SM group under standard medium conditions. However, it would appear that even a stand-alone bSISPCL/HAp scaffold has a strong osteogenic effect on BM-MSCs, without the use of differentiation agents (Fig. 7). The level of differentiation, in terms of ALP expression, in the SM group is close to that of the OM group, in which osteogenic agents (dexamethasone, βglycerophosphate and ascorbic acid) are used.

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ACCEPTED MANUSCRIPT The second marker selected for IF analyses is the Osteopontin (secreted phosphoprotein 1), which is an important bone matrix protein synthesized by both preosteoblastic cells and mature osteoblasts. Its low-phosphorylated 55 kDa form, expressed in the early stages of ossification, supports the formation of a “cement” matrix before bone deposition. Moreover, its highly phosphorylated 44-kDa form, which is expressed by mature

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osteoblasts in subsequent stages, is associated with bone modeling and resorption [59,60].

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OPN is generally used as an early stage osteogenic differentiation marker.

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Fig. 8 shows the IF micrographs of OPN-stained BM-MSC-laden bSIS-PCL/HAp scaffolds. It is evident from the figure that the OPN staining level of the constructs gradually

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increased during the 21-day culture period in both the SM and the OM groups. The highest OPN expression was observed on the 21st day of culture. Nevertheless, the expression of

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OPN observed in the SM group lacking osteogenic inducers was close to, but slightly lower than that observed in the group cultured in the osteogenic differentiation medium.

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Another marker used in the IF analyses is the Osteocalcin, a polypeptide hormone

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specific to osteoblasts, and is associated with the glucose and energy metabolism [61]. OSC is used as a bone turnover marker and a late-stage osteogenic differentiation marker [62,63].

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This protein is expressed in bone formation phases, participates in osteoid mineralization, and is involved in the bone ECM [62,64]. The IF findings demonstrated that (Fig. 9) BM-MSCs

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on bSIS-PCL/HAp expressed OSC, the HAp-binding protein [65], both under the SM and OM conditions, with a time-dependent increase. It is known that, bone ECM affects the differentiation process of the MSCs through both cell-ECM interactions and growth factor activity modulations, in vivo [66]. The fact that this late-stage osteogenic differentiation marker is clearly expressed even under in vitro standard medium conditions suggests that the composite construct containing HAp and bSIS-ECM has the potential to be an effective osteogenic scaffold. 15

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4. Conclusion In this study, a biocompatible osteoinductive multi-layer tissue engineering scaffold was developed using the bovine SIS-ECM, which resembles the bioactive organic content of the natural bone, and with HAp as the inorganic component. The findings show that BM-

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MSCs seeded on the composite bSIS-PCL/HAp scaffold can differentiate in vitro into

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osteogenic cells expressing the late-stage osteogenic markers within 21 days. Future in vivo

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studies involving bSIS-PCL/HAp with "stand-alone” osteogenic effects may reveal the contribution potential of cell-laden and non-cell seeded forms of this scaffold to the new bone

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formation processes.

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Acknowledgements

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Turkey (Grant No. 215M338).

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This work was funded by The Scientific and Technological Research Council of

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ACCEPTED MANUSCRIPT Figure Legends

Fig. 1. Schematic representation of the construction of the multi-layered bSIS-PCL/HAp composite scaffold. (a) Cutting of circular bSIS layers. (b) Stacking of 50 layers using adhesive PCL microdroplets. (c) Perforation of the multilayer construct. (d) Incorporation of

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HAp microparticles.

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Fig. 2. Scanning electron micrographs of the multi-layered bSIS-PCL/HAp composite scaffold. (a) Stacked bSIS-ECM layers are regularly spaced with gaps. Note that PCL does

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not spread along each SIS layer while holding the layers together. (b,c) HAp microparticles are homogenously distributed throughout the construct, and even present in the sectioned

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SEM samples, and in the parts where holes are drilled (indicated with arrows). (d) 30 µm-size

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spherical HAp microparticle on the bSIS layer.

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Fig. 3. Immunophenotypical characterization of BM-MSCs used for the seeding and cell culture on the SIS-PCL/HAp scaffolds. BM-MSCs express high levels of CD29, CD54,

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CD73, CD90 and CD105, but do not express CD31, CD34 or CD45 markers.

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Fig. 4. Bone marrow mesenchymal stem cell growth on multilayer bSIS-PCL/HAp composite scaffold and on standard culture (control) surface. (a) Macroscopic images demonstrating gradual accumulation of the purple-colored formazan crystals on the constructs by time. (b) Spectrophotometric MTT findings retrieved at 1, 3, 7, and 14 days of culture (n = 3 for each group). In the groups, significant differences (p < 0.05) in the mitochondrial dehydrogenase activity are evident after 14 days compared to that in Days 1, 3 and 7.

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ACCEPTED MANUSCRIPT Fig. 5. Scanning electron micrographs of BM-MSC-laden multi-layered bSIS-PCL/HAp composite scaffolds. (a) The bSIS layers are hold together in lamelar form after 14 days of culture. (b-f) HAp microparticles are visible throughout the construct, and even at construct perforations. (g) Mesenchymal stem cells have spread on the layers of the construct, as well as (h) some others have positioned in between neighboring bSIS layers through their cellular

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extensions and ECM (arrows: proliferating cells with pseudopods; *: HAp microparticles; p:

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perforations).

Fig. 6 Mechanical properties of cell–free and BM-MSC-laden bSIS-PCL/HAp composite

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scaffolds. (a) Maximum force and maximum stress values of newly prepared lyophilized and wet cell-free constructs. (b) Mechanical properties of cell-laden constructs under expansion

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culture (SM) and osteogenic differentiation culture (OM) conditions after 21 days (n = 3 for each group). Significant differences are evident between wet and lyophilized constructs (a), as

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well as between the SM and OM groups (b) (p < 0.05).

Fig. 7. Alkaline phosphatase staining of BM-MSC-laden bSIS-PCL/HAp composite scaffolds.

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SM: standard culture; OM: osteogenic differentiation culture. Scale-bars = 200 µm.

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Fig. 8. Osteopontin staining of BM-MSC-laden bSIS-PCL/HAp composite scaffolds. Cells are indicated by arrows. SM: standard culture; OM: osteogenic differentiation culture. Scalebars = 200 µm.

Fig. 9. Osteocalcin staining of BM-MSC-laden bSIS-PCL/HAp composite scaffolds. Cells are indicated by arrows. SM: standard culture; OM: osteogenic differentiation culture. Scale-bars = 200 µm. 25

ACCEPTED MANUSCRIPT Highlights 

A multilayer composite scaffold was developed by assembling decellularized bSIS layers, HAp microparticles and PCL as the binder.

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Mesenchymal stem cells attached to the scaffold differentiated into the osteogenic lineage, by the culture-time-dependent increase in osteogenic markers’ expression.

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Osteoinductive effect can be achieved even without the use of any external

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differentiation inducers.

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